Effects of oxygen provision on the physiology of baker's yeast Saccharomyces cerevisiae

The availability of oxygen has a major effect on all organisms. The yeast Saccharomyces cerevisiae is able to adapt its metabolism for growth in different conditions of oxygen provision, and to grow even under complete lack of oxygen. Although the physiology of S. cerevisiae has mainly been studied under fully aerobic and anaerobic conditions, less is known of metabolism under oxygen-limited conditions and of the adaptation to changing conditions of oxygen provision. This study compared the physiology of S. cerevisiae in conditions of five levels of oxygen provision (0, 0.5, 1.0, 2.8 and 20.9% O2 in feed gas) by using measurements on metabolite, transcriptome and proteome levels. On the transcriptional level, the main differences were observed between the three level groups, 0, 0.5 2.8 and 20.9% O2 which led to fully fermentative, respiro-fermentative and fully respiratory modes of metabolism, respectively. However, proteome analysis suggested post-transcriptional regulation at the level of 0.5 O2. The analysis of metabolite and transcript levels of central carbon metabolism also suggested post-transcriptional regulation especially in glycolysis. Further, a global upregulation of genes related to respiratory pathways was observed in the oxygen-limited conditions and the same trend was seen in the proteome analysis and in the activities of enzymes of the TCA cycle. The responses of intracellular metabolites related to central carbon metabolism and transcriptional responses to change in oxygen availability were studied. As a response to sudden oxygen depletion, concentrations of the metabolites of central carbon metabolism responded faster than the corresponding levels of gene expression. In general, the genome-wide transcriptional responses to oxygen depletion were highly similar when two different initial conditions of oxygen provision (20.9 and 1.0% O2) were compared. The genes related to growth and cell proliferation were transiently downregulated whereas the genes related to protein degradation and phosphate uptake were transiently upregulated. In the cultures initially receiving 1.0% O2, a transient upregulation of genes related to fatty acid oxidation, peroxisomal biogenesis, response to oxidative stress and pentose phosphate pathway was observed. Additionally, this work analysed the effect of oxygen on transcription of genes belonging to the hexose transporter gene family. Although the specific glucose uptake rate was highest in fully anaerobic conditions, none of the hxt genes showed highest expression in anaerobic conditions. However, the expression of genes encoding the moderately low affinity transporters decreased with the decreasing oxygen level. Thus it was concluded that there is a relative increase in high affinity transport in anaerobic conditions supporting the high uptake rate.

U12-type Spliceosome: Localization and Effects of Splicing Efficiency on Gene Expression

The removal of non-coding sequences, introns, is an essential part of messenger RNA processing. In most metazoan organisms, the U12-type spliceosome processes a subset of introns containing highly conserved recognition sequences. U12-type introns constitute less than 0,5% of all introns and reside preferentially in genes related to information processing functions, as opposed to genes encoding for metabolic enzymes. It has previously been shown that the excision of U12-type introns is inefficient compared to that of U2-type introns, supporting the model that these introns could provide a rate-limiting control for gene expression. The low efficiency of U12-type splicing is believed to have important consequences to gene expression by limiting the production of mature mRNAs from genes containing U12-type introns. The inefficiency of U12-type splicing has been attributed to the low abundance of the components of the U12-type spliceosome in cells, but this hypothesis has not been proven. The aim of the first part of this work was to study the effect of the abundance of the spliceosomal snRNA components on splicing. Cells with a low abundance of the U12-type spliceosome were found to inefficiently process U12-type introns encoded by a transfected construct, but the expression levels of endogenous genes were not found to be affected by the abundance of the U12-type spliceosome. However, significant levels of endogenous unspliced U12-type intron-containing pre-mRNAs were detected in cells. Together these results support the idea that U12-type splicing may limit gene expression in some situations. The inefficiency of U12-type splicing has also promoted the idea that the U12-type spliceosome may control gene expression, limiting the mRNA levels of some U12-type intron-containing genes. While the identities of the primary target genes that contain U12-type introns are relatively well known, little has previously been known about the downstream genes and pathways potentially affected by the efficiency of U12-type intron processing. Here, the effects of U12-type splicing efficiency on a whole organism were studied in a Drosophila line with a mutation in an essential U12-type spliceosome component. Genes containing U12-type introns showed variable gene-specific responses to the splicing defect, which points to variation in the susceptibility of different genes to changes in splicing efficiency. Surprisingly, microarray screening revealed that metabolic genes were enriched among downstream effects, and that the phenotype could largely be attributed to one U12-type intron-containing mitochondrial gene. Gene expression control by the U12-type spliceosome could thus have widespread effects on metabolic functions in the organism. The subcellular localization of the U12-type spliceosome components was studied as a response to a recent dispute on the localization of the U12-type spliceosome. All components studied were found to be nuclear indicating that the processing of U12-type introns occurs within the nucleus, thus clarifying a question central to the field. The results suggest that the U12-type spliceosome can limit the expression of genes that contain U12-type introns in a gene-specific manner. Through its limiting role in pre-mRNA processing, the U12-type splicing activity can affect specific genetic pathways, which in the case of Drosophila are involved in metabolic functions.

Effects of management and landscape structure on biodiversity in boreal agricultural farmland

Intensified agricultural practises introduced after the Second World War are identified as a major cause of global biodiversity declines. In several European countries agri-environment support schemes have been introduced to counteract the ongoing biodiversity declines. Farmers participating in agri-environment schemes are financially compensated for decreasing the intensity of farming practises leading to smaller yields and lower income. The Finnish agri-environment support scheme is composed of a set of measures, such as widened field margins along main ditches (obligatory measure), management of features increasing landscape diversity, management of semi-natural grasslands, and organic farming (special agreement measures). The magnitude of the benefits for biodiversity depends on landscape context and the properties of individual schemes. In this thesis I studied whether one agri-environment scheme, organic farming, is beneficial for species diversity and abundance of diurnal lepidopterans, bumblebees, carabid beetles and arable weeds. I found that organic farming did not enhance species richness of selected insect taxa, although bumblebee species richness tended to be higher in organic farms. Abundance of lepidopterans and bumblebees was not enhanced by organic farming, but carabid beetle abundance was higher in mixed farms with both cereal crop production and animal husbandry. Both species richness and abundance of arable weeds were higher in organic farms. My second objective was to study how landscape structure shapes farmland butterfly communities. I found that the percentage of habitat specialists and species with poor dispersal abilities in butterfly assemblages decreased with increasing arable field cover, leading to a dramatic decrease in butterfly beta diversity. In field boundaries local species richness of butterflies was linearly related to landscape species richness in geographic regions with high arable field cover, indicating that butterfly species richness in field boundaries is more limited by landscape factors than local habitat factors. In study landscapes containing semi-natural grasslands the relationship decelerated at high landscape species richness, suggesting that local species richness of butterflies in field boundaries is limited by habitat factors (demanding habitat specialists that occurred in semi-natural grasslands were absent in field margins). My results suggest that management options in field margins will affect mainly generalists, and species with good dispersal abilities, in landscapes with high arable field cover. Habitat specialists and species with poor dispersal abilities may benefit of management options if these are applied in the vicinity of source populations.

Calculations of the free energy of interaction of the c-Fos−c-Jun coiled coil: Effects of the solvation model and the inclusion of polarization effects

The leucine zipper region of activator protein-1 (AP-1) comprises the c-Jun and c-Fos proteins and constitutes a well-known coiled coil protein−protein interaction motif. We have used molecular dynamics (MD) simulations in conjunction with the molecular mechanics/Poisson−Boltzmann generalized-Born surface area [MM/PB(GB)SA] methods to predict the free energy of interaction of these proteins. In particular, the influence of the choice of solvation model, protein force field, and water potential on the stability and dynamic properties of the c-Fos−c-Jun complex were investigated. Use of the AMBER polarizable force field ff02 in combination with the polarizable POL3 water potential was found to result in increased stability of the c-Fos−c-Jun complex. MM/PB(GB)SA calculations revealed that MD simulations using the POL3 water potential give the lowest predicted free energies of interaction compared to other nonpolarizable water potentials. In addition, the calculated absolute free energy of binding was predicted to be closest to the experimental value using the MM/GBSA method with independent MD simulation trajectories using the POL3 water potential and the polarizable ff02 force field, while all other binding affinities were overestimated.

A comparative study of the early effects of phenobarbital and 3-methylcholanthrene on the synthesis and transport of ribonucleic acid in rat liver.

At 2-3 h after phenobaribtal administration, the drug has no effect on nucleoplasmic RNA synthesis and decreases nucleolar RNA synthesis. However, at this time there is an increase in the labelling of cytoplasmic poly(A)-containing RNA, even though there is decreased labelling of total polyribosomal RNA. The decrease in labelling of nucleolar and total polyribosomal RNA owing to phenobarbital is a transient phenomenon. Under similar conditions, 3-methylcholanthrene has no effect on nucleolar RNA synthesis, but leads to an increase in synthesis of nucleoplasmic and cytoplasmic poly(A)-containing RNA. Cytosol isolated from phenobarbital-treated, but not from 3-methyl-cholanthrene-treated, animals facilitates an enhanced transport of RNA from nuclei. At the time points investigated, 3-methylcholanthrene or its metabolite shows a 10-15-fold higher concentration in the chromatin than that of phenobarbital or its metabolite. It is suggested that the primary effect of phenobarbital is at the cytoplasmic level, promoting the transport of RNA from the nuclei, which can act as a trigger for enhanced transcription at later periods. 3-Methylcholanthrene or its metabolite directly binds to the chromatin and evokes a selective transcriptional response.

The effects of eutrophication on alternative reproductive tactics in threespine sticklebacks

When organisms compete for mates and fertilisations, the process of sexual selection drives the evolution of traits that increase reproductive success. The traits targeted by selection, and the extent to which they change, are constrained by the local environment. Sexual selection due to female mate choice can be undermined by alternative reproductive tactics (ARTs), which refers to discontinuous variation in traits or behaviours used in reproduction. As human activities are rapidly changing our planet, this raises the question how ARTs will be affected. Fish show a bewildering diversity of ARTs, which make them good model organisms to answer these questions. One example of human-induced environmental change, which is affecting aquatic ecosystems around the world, is eutrophication, the over-enrichment of water bodies with nutrients. One of its effects is decreased underwater visibility due to increases in both turbidity and vegetation density. The aims of this thesis were to investigate the effects increased turbidity and vegetation density have on an ART in sticklebacks, a fish common to marine and fresh water bodies of the Northern hemisphere. I furthermore investigated how this affected sexual selection for male size, a trait commonly under selection. I used a combination of behavioural observations in microcosms, where I manipulated underwater visibility, with collection of genetic material to reconstruct parentage of broods, and thus identify sneak fertilisations. The results show that turbidity might have weak negative effects on the frequency of sneaking behaviour, although this behaviour was rather infrequent in these experiments, which complicates firm conclusions. In dense vegetation the number of sneak fertilisations decreased slightly, as fewer nesting males sneaked, while the number of non-nesting males sneaking remained constant. The paternity analyses revealed that a significantly smaller fraction of eggs was sneak fertilised under dense vegetation. Furthermore, amongst the nesting males that sneaked, the amount of eggs sneak fertilised correlated positively with courtship success. A reduction in sneaking by these males under dense vegetation equalised the distribution of fertilisation success, in turn contributing to a decrease in the opportunity for selection. Under dense vegetation significantly more males built nests, which has also been observed in previous field studies. In a separate experiment we addressed if such changes in the proportion of nesters and non-nesters, without changes in visibility, affected the incidence of sneak fertilisation. My results show this was not the case, likely because sneaking is an opportunistic tactic shown by both nesters and non-nesters. Non-nesters did sneak proportionately more when there were many of them, which could be due to changes in the cost-benefit ratio of sneaking. As nesters can only attack one intruder at a time, the costs and risks per sneaker will decrease as the number of sneakers increases. The defensive behaviours shown by the nesters before spawning shifted to a more aggressive form of nest defence. This could be because less aggressive behaviours lose their effectiveness when the number of intruders increases. It could also indicate that the risks associated with aggressive behaviours decrease when there are fewer fellow nesters, as other studies indicate nesters are competitive and aggressive individuals. Under turbid conditions I did not detect changes in the opportunity for selection, based on fertilisation success, nor was male size under significant selection under clear or turbid conditions. More thorough analyses under densely vegetated conditions across the nesting, courtship and fertilisation stages revealed a decrease in the opportunity for selection across all stages. A reduction in sneaking by nesters contributed to this. During the nesting stage, but not during later stages, body size was under significant directional selection under sparse, but not dense vegetation. This illustrates the importance of considering all selection stages to get a complete picture of how environmental changes affect sexual selection. Leaving out certain stages or subgroups can result in incomplete or misleading results.

Effects of transit quality of service characteristics on daily bus ridership

A case study of Brisbane, the capital city of Queensland, Australia, explored how explicit measures of transit quality of service (e.g., service frequency, service span, and travel time ratio) and implicit environmental predictors (e.g., topographic grade factor) influenced bus ridership. The primary hypothesis tested was that bus ridership was higher in suburbs with high transit quality of service than in suburbs with limited service quality. Multiple linear regression, used to identify a strong positive relationship between route intensity (bus-km/h-km2) and bus ridership, indicated that both increased service frequency and spatial route density corresponded to higher bus ridership. Additionally, the travel time ratio (i.e., the ratio of in-vehicle transit travel time to in-vehicle automobile travel time) had a significant negative association with suburban ridership: transit use declined as travel time ratio increased. In contrast, topographic grade and service span did not significantly affect suburban bus ridership. The study findings enhance the fundamental understanding of traveler behavior, which is informative to urban transportation policy, planning, and provision.

Public good provision and the welfare effects of indirect tax harmonisation

The paper considers the welfare effect of the harmonisation of indirect taxes in two open economies. The revenue from taxation is used for the production of a non-tradeable public good. The welfare levels are affected via two channels: (i) changes in the levels of public good provision, and (ii) changes in deadweight loss associated with the taxes. We develop a number of rules of harmonisation and derive conditions under which they lead to potential Pareto improvement.

Effects of mould filling on evolution of the solid-liquid interface during solidification

This work presents a numerical analysis of simultaneous mould filling and phase change for solidification in a two-dimensional rectangular cavity. The role of residual flow strength and temperature gradients within the solidifying domain, caused by the filling process, on the evolution of solidification interface are investigated. An implicit volume of fluid (VOF)-based algorithm has been employed for simulating the free surface flows during the filling process, while the model for solidification is based on a fixed-grid enthalpy-based control volume approach. Solidification modeling is coupled with VOF through User Defined Functions developed in the commercial computational fluid dynamics (CFD) code FLUENT 6.3.26. Comparison between results of the conventional analysis without filling effect and those of the present analysis shows that the residual flow resulting from the filling process significantly influences the progress of the solidification interface. A parametric study is also performed with variables such as cooling rate, filling velocity and filling configuration, in order to investigate the coupled effects of the buoyancy-driven flow and the residual flow on the solidification behavior.

The non-linear response of a gyrostabilized platform: Effects of correction torque time delay and of random inputs

First, the non-linear response of a gyrostabilized platform to a small constant input torque is analyzed in respect to the effect of the time delay (inherent or deliberately introduced) in the correction torque supplied by the servomotor, which itself may be non-linear to a certain extent. The equation of motion of the platform system is a third order nonlinear non-homogeneous differential equation. An approximate analytical method of solution of this equation is utilized. The value of the delay at which the platform response becomes unstable has been calculated by using this approximate analytical method. The procedure is illustrated by means of a numerical example. Second, the non-linear response of the platform to a random input has been obtained. The effects of several types of non-linearity on reducing the level of the mean square response have been investigated, by applying the technique of equivalent linearization and solving the resulting integral equations by using laguerre or Gaussian integration techniques. The mean square responses to white noise and band limited white noise, for various values of the non-linear parameter and for different types of non-linearity function, have been obtained. For positive values of the non-linear parameter the levels of the non-linear mean square responses to both white noise and band-limited white noise are low as compared to the linear mean square response. For negative values of the non-linear parameter the level of the non-linear mean square response at first increases slowly with increasing values of the non-linear parameter and then suddenly jumps to a high level, at a certain value of the non-linearity parameter.