Transcriptome and proteome analysis of xylose-metabolising Saccharomyces cerevisiae

Increasing concern about global climate warming has accelerated research into renewable energy sources that could replace fossil petroleum-based fuels and materials. Bioethanol production from cellulosic biomass by fermentation with baker s yeast Saccharomyces cerevisiae is one of the most studied areas in this field. The focus has been on metabolic engineering of S. cerevisiae for utilisation of the pentose sugars, in particular D-xylose that is abundant in the hemicellulose fraction of biomass. Introduction of a heterologous xylose-utilisation pathway into S. cerevisiae enables xylose fermentation, but ethanol yield and productivity do not reach the theoretical level. In the present study, transcription, proteome and metabolic flux analyses of recombinant xylose-utilising S. cerevisiae expressing the genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis and the endogenous xylulokinase were carried out to characterise the global cellular responses to metabolism of xylose. The aim of these studies was to find novel ways to engineer cells for improved xylose fermentation. The analyses were carried out from cells grown on xylose and glucose both in batch and chemostat cultures. A particularly interesting observation was that several proteins had post-translationally modified forms with different abundance in cells grown on xylose and glucose. Hexokinase 2, glucokinase and both enolase isoenzymes 1 and 2 were phosphorylated differently on the two different carbon sources studied. This suggests that phosphorylation of glycolytic enzymes may be a yet poorly understood means to modulate their activity or function. The results also showed that metabolism of xylose affected the gene expression and abundance of proteins in pathways leading to acetyl-CoA synthesis and altered the metabolic fluxes in these pathways. Additionally, the analyses showed increased expression and abundance of several other genes and proteins involved in cellular redox reactions (e.g. aldo-ketoreductase Gcy1p and 6-phosphogluconate dehydrogenase) in cells grown on xylose. Metabolic flux analysis indicated increased NADPH-generating flux through the oxidative part of the pentose phosphate pathway in cells grown on xylose. The most importantly, results indicated that xylose was not able to repress to the same extent as glucose the genes of the tricarboxylic acid and glyoxylate cycles, gluconeogenesis and some other genes involved in the metabolism of respiratory carbon sources. This suggests that xylose is not recognised as a fully fermentative carbon source by the recombinant S. cerevisiae that may be one of the major reasons for the suboptimal fermentation of xylose. The regulatory network for carbon source recognition and catabolite repression is complex and its functions are only partly known. Consequently, multiple genetic modifications and also random approaches would probably be required if these pathways were to be modified for further improvement of xylose fermentation by recombinant S. cerevisiae strains.

Impacts of agriculture on amphibians at multiple scales

Agriculture-mediated habitat loss and degradation together with climate change are among the greatest global threats to species, communities, and ecosystem functioning. During the last century, more than 50% of the world's wetlands have been lost and agricultural activities have subjected wetland species to increased isolation and decreased quality of habitats. Likewise, as a part of agricultural intensification, the use of pesticides has increased notably, and pesticide residues occur frequently in wetlands making the exposure of wetland organisms to pesticides highly probable. In this thesis, a set of ecotoxicological and landscape ecological studies were carried out to investigate pesticide-effects on tadpoles, and species-habitat relationships of amphibians in agricultural landscapes. The results show that the fitness of R. temporaria tadpoles can be negatively affected by sublethal pesticide concentrations, and that pesticides may increase the costs of response to natural environmental stressors. However, tadpoles may also be able to compensate for some of the negative effects of pesticides. The results further demonstrate that both historic and current-day agricultural land use can negatively impact amphibians, but that in some cases the costs of living in agricultural habitats may only become apparent when amphibians face other environmental stressors, such as drought. Habitat heterogeneity may, however, increase the persistence of amphibians in agricultural landscapes. Hence, the results suggest that amphibians are likely to be affected by agricultural processes that operate at several spatial and temporal scales, and that it is probable that various processes related to current-day agriculture will affect both larval and adult amphibians. The results imply that maintaining dense wetland patterns could enhance persistence of amphibian populations in agricultural habitats, and indicate that heterogeneous landscapes may lower the risk of regional amphibian population declines under extreme weather perturbations.

Grids with multiple batch systems for performance enhancement of multi-component and parameter sweep parallel applications

In this work, we evaluate the benefits of using Grids with multiple batch systems to improve the performance of multi-component and parameter sweep parallel applications by reduction in queue waiting times. Using different job traces of different loads, job distributions and queue waiting times corresponding to three different queuing policies(FCFS, conservative and EASY backfilling), we conducted a large number of experiments using simulators of two important classes of applications. The first simulator models Community Climate System Model (CCSM), a prominent multi-component application and the second simulator models parameter sweep applications. We compare the performance of the applications when executed on multiple batch systems and on a single batch system for different system and application configurations. We show that there are a large number of configurations for which application execution using multiple batch systems can give improved performance over execution on a single system.

Genetic analysis of chromosomal regions 2q33, 7q32 and 19q13 in multiple sclerosis susceptibility

Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system (CNS) affecting 0.1-0.2% of Northern European descent population. MS is considered to be a multifactorial disease, both environment and genetics play a role in its pathogenesis. Despite several decades of intense research, the etiological and pathogenic mechanisms underlying MS remain still largely unknown and no curative treatment exists. The genetic architecture underlying MS is complex with multiple genes involved. The strongest and the best characterized predisposing genetic factors for MS are located, as in other immune-mediated diseases, in the major histocompatibility complex (MHC) on chromosome 6. In humans MHC is called human leukocyte antigen (HLA). Alleles of the HLA locus have been found to associate strongly with MS and remained for many years the only consistently replicable genetic associations. However, recently other genes located outside the MHC region have been proposed as strong candidates for susceptibility to MS in several studies. In this thesis a new genetic locus located on chromosome 7q32, interferon regulatory factor 5 (IRF5), was identified in the susceptibility to MS. In particular, we found that common variation of the gene was associated with the disease in three different populations, Spanish, Swedish and Finnish. We also suggested a possible functional role for one of the risk alleles with impact on the expression of the IRF5 locus. Previous studies have pointed out a possible role played by chromosome 2q33 in the susceptibility to MS and other autoimmune disorders. The work described here also investigated the involvement of this chromosomal region in MS predisposition. After the detection of genetic association with 2q33 (article-1), we extended our analysis through fine-scale single nucleotide polymorphism (SNP) mapping to define further the contribution of this genomic area to disease pathogenesis (article-4). We found a trend (p=0.04) for association to MS with an intronic SNP located in the inducible T-cell co-stimulator (ICOS) gene, an important player in the co-stimulatory pathway of the immune system. Expression analysis of ICOS revealed a novel, previously uncharacterized, alternatively spliced isoform, lacking the extracellular domain that is needed for ligand binding. The stability of the newly-identified transcript variant and its subcellular localization were analyzed. These studies indicated that the novel isoform is stable and shows different subcellular localization as compared to full-length ICOS. The novel isoform might have a regulatory function, but further studies are required to elucidate its function. Chromosome 19q13 has been previously suggested as one of the genomic areas involved in MS predisposition. In several populations, suggestive linkage signals between MS predisposition and 19q13 have been obtained. Here, we analysed the role of allelic variation in 19q13 by family based association analysis in 782 MS families collected from Finland. In this dataset, we were not able to detect any statistically significant associations, although several previously suggested markers were included to the analysis. Replication of the previous findings on the basis of linkage disequilibrium between marker allele and disease/risk allele appears notoriously difficult because of limitations such as allelic heterogeneity. Re-sequencing based approaches may be required for elucidating the role of chromosome 19q13 with MS. This thesis has resulted in the identification of a new MS susceptibility locus (IRF5) previously associated with other inflammatory or autoimmune disorders, such as SLE. IRF5 is one of the mediators of interferons biological function. In addition to providing new insight in the possible pathogenetic pathway of the disease, this finding suggests that there might be common mechanisms between different immune-mediated disorders. Furthermore the work presented here has uncovered a novel isoform of ICOS, which may play a role in regulatory mechanisms of ICOS, an important mediator of lymphocyte activation. Further work is required to uncover its functions and possible involvement of the ICOS locus in MS susceptibility.

Human mesenchymal stem cells retain multilineage differentiation capacity including neural marker expression after extended in vitro expansion

The suitability of human mesenchymal stem cells (hMSCs) in regenerative medicine relies on retention of their proliferative expansion potential in conjunction with the ability to differentiate toward multiple lineages. Successful utilisation of these cells in clinical applications linked to tissue regeneration requires consideration of biomarker expression, time in culture and donor age, as well as their ability to differentiate towards mesenchymal (bone, cartilage, fat) or non-mesenchymal (e.g., neural) lineages. To identify potential therapeutic suitability we examined hMSCs after extended expansion including morphological changes, potency (stemness) and multilineage potential. Commercially available hMSC populations were expanded in vitro for > 20 passages, equating to > 60 days and > 50 population doublings. Distinct growth phases (A-C) were observed during serial passaging and cells were characterised for stemness and lineage markers at representative stages (Phase A: P+5, approximately 13 days in culture; Phase B: P+7, approximately 20 days in culture; and Phase C: P+13, approximately 43 days in culture). Cell surface markers, stem cell markers and lineage-specific markers were characterised by FACS, ICC and Q-PCR revealing MSCs maintained their multilineage potential, including neural lineages throughout expansion. Co-expression of multiple lineage markers along with continued CD45 expression in MSCs did not affect completion of osteogenic and adipogenic specification or the formation of neurospheres. Improved standardised isolation and characterisation of MSCs may facilitate the identification of biomarkers to improve therapeutic efficacy to ensure increased reproducibility and routine production of MSCs for therapeutic applications including neural repair.

Autonomous greenhouse gas sampling using multiple robotic boats

Accurately quantifying total greenhouse gas emissions (e.g. methane) from natural systems such as lakes, reservoirs and wetlands requires the spatial-temporal measurement of both diffusive and ebullitive (bubbling) emissions. Traditional, manual, measurement techniques provide only limited localised assessment of methane flux, often introducing significant errors when extrapolated to the whole-of-system. In this paper, we directly address these current sampling limitations and present a novel multiple robotic boat system configured to measure the spatiotemporal release of methane to atmosphere across inland waterways. The system, consisting of multiple networked Autonomous Surface Vehicles (ASVs) and capable of persistent operation, enables scientists to remotely evaluate the performance of sampling and modelling algorithms for real-world process quantification over extended periods of time. This paper provides an overview of the multi-robot sampling system including the vehicle and gas sampling unit design. Experimental results are shown demonstrating the system’s ability to autonomously navigate and implement an exploratory sampling algorithm to measure methane emissions on two inland reservoirs.

A Joint Source-Channel Coding Scheme for Transmission of Correlated Discrete Sources over a Gaussian Multiple Access Channel

We consider the problem of transmission of correlated discrete alphabet sources over a Gaussian Multiple Access Channel (GMAC). A distributed bit-to-Gaussian mapping is proposed which yields jointly Gaussian codewords. This can guarantee lossless transmission or lossy transmission with given distortions, if possible. The technique can be extended to the system with side information at the encoders and decoder.

Genome-wide DNA methylation profiling of CD8+ T cells shows a distinct epigenetic signature to CD4+ T cells in multiple sclerosis patients

Background Multiple sclerosis (MS) is thought to be a T cell-mediated autoimmune disorder. MS pathogenesis is likely due to a genetic predisposition triggered by a variety of environmental factors. Epigenetics, particularly DNA methylation, provide a logical interface for environmental factors to influence the genome. In this study we aim to identify DNA methylation changes associated with MS in CD8+ T cells in 30 relapsing remitting MS patients and 28 healthy blood donors using Illumina 450K methylation arrays. Findings Seventy-nine differentially methylated CpGs were associated with MS. The methylation profile of CD8+ T cells was distinctive from our previously published data on CD4+ T cells in the same cohort. Most notably, there was no major CpG effect at the MS risk gene HLA-DRB1 locus in the CD8+ T cells. Conclusion CD8+ T cells and CD4+ T cells have distinct DNA methylation profiles. This case–control study highlights the importance of distinctive cell subtypes when investigating epigenetic changes in MS and other complex diseases.

Conservative Design Optimization of Laminated Composite Structures Using Genetic Algorithms and Multiple Failure Criteria

This article analyzes the effect of devising a new failure envelope by the combination of the most commonly used failure criteria for the composite laminates, on the design of composite structures. The failure criteria considered for the study are maximum stress and Tsai-Wu criteria. In addition to these popular phenomenological-based failure criteria, a micromechanics-based failure criterion called failure mechanism-based failure criterion is also considered. The failure envelopes obtained by these failure criteria are superimposed over one another and a new failure envelope is constructed based on the lowest absolute values of the strengths predicted by these failure criteria. Thus, the new failure envelope so obtained is named as most conservative failure envelope. A minimum weight design of composite laminates is performed using genetic algorithms. In addition to this, the effect of stacking sequence on the minimum weight of the laminate is also studied. Results are compared for the different failure envelopes and the conservative design is evaluated, with respect to the designs obtained by using only one failure criteria. The design approach is recommended for structures where composites are the key load-carrying members such as helicopter rotor blades.

Power dissipation for systems with junctions of multiple quantum wires

We study power dissipation for systems of multiple quantum wires meeting at a junction, in terms of a current splitting matrix (M) describing the junction. We present a unified framework for studying dissipation for wires with either interacting electrons (i.e., Tomonaga-Luttinger liquid wires with Fermi-liquid leads) or noninteracting electrons. We show that for a given matrix M, the eigenvalues of (MM)-M-T characterize the dissipation, and the eigenvectors identify the combinations of bias voltages which need to be applied to the different wires in order to maximize the dissipation associated with the junction. We use our analysis to propose and study some microscopic models of a dissipative junction which employ the edge states of a quantum Hall liquid. These models realize some specific forms of the M matrix whose entries depends on the tunneling amplitudes between the different edges.

A Kalman filter based strategy for linear structural system identification based on multiple static and dynamic test data

The problem of identification of stiffness, mass and damping properties of linear structural systems, based on multiple sets of measurement data originating from static and dynamic tests is considered. A strategy, within the framework of Kalman filter based dynamic state estimation, is proposed to tackle this problem. The static tests consists of measurement of response of the structure to slowly moving loads, and to static loads whose magnitude are varied incrementally; the dynamic tests involve measurement of a few elements of the frequency response function (FRF) matrix. These measurements are taken to be contaminated by additive Gaussian noise. An artificial independent variable τ, that simultaneously parameterizes the point of application of the moving load, the magnitude of the incrementally varied static load and the driving frequency in the FRFs, is introduced. The state vector is taken to consist of system parameters to be identified. The fact that these parameters are independent of the variable τ is taken to constitute the set of ‘process’ equations. The measurement equations are derived based on the mechanics of the problem and, quantities, such as displacements and/or strains, are taken to be measured. A recursive algorithm that employs a linearization strategy based on Neumann’s expansion of structural static and dynamic stiffness matrices, and, which provides posterior estimates of the mean and covariance of the unknown system parameters, is developed. The satisfactory performance of the proposed approach is illustrated by considering the problem of the identification of the dynamic properties of an inhomogeneous beam and the axial rigidities of members of a truss structure.

Analysis of the MAP/G(a,b)/1/N queue with multiple vacations

This paper deals with a batch service queue and multiple vacations. The system consists of a single server and a waiting room of finite capacity. Arrival of customers follows a Markovian arrival process (MAP). The server is unavailable for occasional intervals of time called vacations, and when it is available, customers are served in batches of maximum size ‘b’ with a minimum threshold value ‘a’. We obtain the queue length distributions at various epochs along with some key performance measures. Finally, some numerical results have been presented.

Absolute quantification of human tear lactoferrin using multiple reaction monitoring technique with stable-isotopic labeling

The mass spectrometry technique of multiple reaction monitoring (MRM) was used to quantify and compare the expression level of lactoferrin in tear films among control, prostate cancer (CaP), and benign prostate hyperplasia (BPH) groups. Tear samples from 14 men with CaP, 15 men with BPH, and 14 controls were analyzed in the study. Collected tears (2 μl) of each sample were digested with trypsin overnight at 37 °C without any pretreatment, and tear lactoferrin was quantified using a lactoferrin-specific peptide, VPSHAVVAR, both using natural/light and isotopic-labeled/heavy peptides with MRM. The average tear lactoferrin concentration was 1.01 ± 0.07 μg/μl in control samples, 0.96 ± 0.07 μg/μl in the BPH group, and 0.98 ± 0.07 μg/μl in the CaP group. Our study is the first to quantify tear proteins using a total of 43 individual (non-pooled) tear samples and showed that direct digestion of tear samples is suitable for MRM studies. The calculated average lactoferrin concentration in the control group matched that in the published range of human tear lactoferrin concentration measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the lactoferrin was stably expressed across all of the samples, with no significant differences being observed among the control, BPH, and CaP groups.

Characterization of anomalous relaxation using the time-fractional Bloch equation and multiple echo T2 *-weighted magnetic resonance imaging at 7T

PURPOSE To study the utility of fractional calculus in modeling gradient-recalled echo MRI signal decay in the normal human brain. METHODS We solved analytically the extended time-fractional Bloch equations resulting in five model parameters, namely, the amplitude, relaxation rate, order of the time-fractional derivative, frequency shift, and constant offset. Voxel-level temporal fitting of the MRI signal was performed using the classical monoexponential model, a previously developed anomalous relaxation model, and using our extended time-fractional relaxation model. Nine brain regions segmented from multiple echo gradient-recalled echo 7 Tesla MRI data acquired from five participants were then used to investigate the characteristics of the extended time-fractional model parameters. RESULTS We found that the extended time-fractional model is able to fit the experimental data with smaller mean squared error than the classical monoexponential relaxation model and the anomalous relaxation model, which do not account for frequency shift. CONCLUSIONS We were able to fit multiple echo time MRI data with high accuracy using the developed model. Parameters of the model likely capture information on microstructural and susceptibility-induced changes in the human brain.