Browsing by discipline "Bioteknik (MAAT)"

In Norway spruce (Picea abies L. Karst.) lignin forms a major part of the xylem cell walls and renders the tracheids water resistant while giving support to the cell walls and the whole tree trunk. In Norway spruce lignin is polymerized mainly from coniferyl alcohol but the origins of this monolignol are not known. In our study, we employed laser capture microdissection (LCMD) system to isolate ray parenchyma cells and xylem tracheids from thin (30-40 μm), tangential cryomicrotome sections of developing xylem of the spruce trunk. We wanted to analyse the monolignol biosynthesis pathway gene expression separately in these cell types. Our aim was to examine the possibility that coniferyl alcohol is produced in the tracheid cells, or whether also the neighboring cells (ray cells) contribute to the biosynthesis of monolignols during the lignification as has been confirmed in angiosperms before. Total RNA extracted from the collected material was used to perform low mRNA input sequencing on the Illumina HiSeq platform to identify transcripts potentially involved in monolignol biosynthesis and secretion. As a control material we used whole cryomicrotome sections containing both xylem tracheids and rays. Deep sequencing was performed to generate quantitative expression data within a particular cell type. Differential gene expression was conducted with a Chipster analysis software by using DESeq2. Altogether, 936 genes were differentially expressed between whole sections and ray cells, and 424 transcripts were more abundant in ray cells, while 512 transcripts were less abundant. Comparison between whole sections and xylem tracheids illustrated that 18 transcripts were more abundant in xylem tracheids and 275 transcripts were less abundant. Our study demonstrated the usefulness of LCMD combined with RNA-Seq to characterize gene expression in specific cell types.

Efficient utilization of renewable plant and crop based biomass is one of the main areas of study in industrial biotechnology. Roughly 20-30 % of all plant biomass consists of hemicellulose, a polymer composed of six carbon (hexose) and five carbon (pentose) sugars. This proves a challenge in utilizing all available plant biomass efficiently, since many micro-organisms, which for instance readily ferment glucose (a hexose) into ethanol, will not do so for pentose sugars. Alternative pathways for utilization of pentose sugars are being looked for. Characterization of the Caulobacter crescentus D-xylonate dehydratase (Cc XyDHT) was the aim of this study. Cc XyDHT belongs to the ILVD/EDD protein family, whose members incorporate an iron-sulfur (FeS) cluster into their protein structure coordinated by two to four cysteine amino acids. Removal of the cysteine ligands should disrupt the incorporation of the FeS cluster, and thus presumably the activity of the enzyme. Three cysteines in Cc XyDHT which could act as ligands for the FeS cluster had previously been identified by sequence alignment among the ILVD/EDD family. Accordingly, four single cysteine-to-serine mutants of the Cc XyDHT as expression plasmid constructs were designed, with a non-conserved cysteine residue selected as a control. The wild type and mutated Cc XyDHTs were produced in Escherichia coli and purified with affinity chromatography using Strep-tag. SDS-page and Western blotting with an anti-Strep-tag antibody were used to confirm that the expressed proteins were Cc XyDHTs. The mutations’ effects on the protein fold and to the presence of the FeS cluster were investigated with UV and circular dichroism spectroscopy. Cc XyDHT catalyzes the conversion of D-xylonate into 2-keto-3-deoxy-xylonate. A colorimetric assay using thiobarbituric acid (TBA) as the reactant was used to measure enzymatic activity. The pH and temperature optima, substrate specificity, and enzyme kinetics of the wild type Cc XyDHT were determined. For the two best substrates, kcat/Km = 1220 mM/min for D-xylonate and kcat/Km = 1160 mM/min for D-gluconate were found. All three conserved mutations were found to reduce enzymatic activity more than 99 % with these substrates. Depending on which cysteine was disrupted, differences in the kinetic constants between D-xylonate and D-gluconate were found. The non-conserved mutation reduced activity by approx. 40 %. The spectroscopic results indicate that the three conserved mutants lacked the FeS cluster, while the non-conserved mutant still incorporated it. It can be concluded that the three conserved cysteines are involved in coordinating the FeS cluster, which itself plays an important role in the catalytic activity of Cc XyDHT. These findings should be helpful for follow-up studies in biotechnological application of pentose sugars acids and their derivatives.

Selective degeneration and dysregulation of specific neuronal populations is a common hallmark shared by neurodegenerative diseases affecting the aging population. Parkinson’s disease (PD) is one of the most prevalent neurodegenerative diseases with debilitating clinical manifestations that follow a chronic and progressive course. Pathological hallmarks of PD involve gradual and specific loss of DA (DA) neurons and widespread presence of Lewy body (LB) inclusions that consist of aggregated presynaptic protein, α-Synuclein (αSyn). Treatment of PD remains to be at symptomatic management as the underlying mechanisms that trigger neurodegeneration are still not fully elucidated. Over the past two decades, microRNAs (miRNAs) have become a major area of interest within biomedical fields and gained increasing momentum in the context of neurodegenerative diseases. In recent developments, changes in mature miRNA profiles have been reported in aging tissue and many age-related diseases, including PD. More recently, a number of studies have found that the most essential enzyme in the miRNA biogenesis pathway, Dicer, exhibits reduced expression with aging. To these ends, a genetic mouse model based on heterozygous knockout of Dicer (DicerHET) was introduced to simulate Dicer downregulation. Initial investigations identified the DicerHET model as a promising tool for studying the relationship between disrupted miRNA biogenesis and neurodegeneration associated with PD. To facilitate future investigations and speed up screening of potential therapeutic compounds using this genetic model, in the current work, we aimed to produce a DicerHET in vitro model with a practical and convenient genetic engineering approach. The main focus of this work was to validate the model and establish a standardized reproducible approach suitable for research that addresses the role of miRNA biogenesis in PD. The desired DicerHET genotype was generated in vitro by employing traditional Cre/loxP system in conjunction with a virally mediated Cre expression. More specifically, primary cortical cultures, derived from Dicer flox/+ mice embryos, were transduced with Cre expressing lentiviral vectors (lenti-hSYN-T2A-Cre) to delete the “floxed” Dicer allele. To establish optimal parameters for the procedure, we analysed recombination efficiency under different transduction conditions. The most efficient recombination was achieved after 5 days of induction in cultures. However, we observed that DicerHET genotype did not attenuate survival of the cells, as assessed by immunohistochemistry. Further, as a proof of concept, we exposed the DicerHET cultures to pre-formed fibrils (PFFs) - a PD related stressor that causes αSyn aggregation. pSer129-αSyn-positive LB-like aggregates were detected in all the PFF-treated cultures, however, with a greater accumulation in the DicerHET cultures. Interestingly, increased aggregation was not accompanied by increased cell death, suggesting that DicerHET genotype does not increase vulnerability of cortical neurons to pSer129-αSyn aggregation. Based on our earlier studies we presume that DA neurons may bear a specific vulnerability towards the age-related Dicer depletion. More conclusive evidence on this intriguing relationship could be provided in future research using the DicerHET model that can be readily applied to primary DA cultures.

Metabolic disorders are known to predispose dairy cows to periparturient diseases. Main components of metabolic disorder are insulin resistance and severe negative energy balance which are responsible for the reduced fertility in addition of increased risk of disease in dairy cows. The mobilization of adipose tissue in response to energy deficiency is associated with metabolic and endocrine changes during early lactation. Reduced insulin sensitivity in peripheral tissues could potentially change the relative rates of lipolysis and lipogenesis. This study focused on expression of lipogenesis and lipolysis associated genes around parturition. Samples and data sets for this study were obtained from the feeding experiments conducted from September 2010 to April 2011 in Viikki Experimental Farm at the University of Helsinki. Sixteen multiparous Ayrshire cows were divided into two groups based on energy level and fibre content of their feed: (1) grass silage group (control) and (2) silage-roughage mixture group (experiment). During experimental period average energy intake (MJ/day) in silage group was 35% higher than in silage-roughage mixture group. Subcutaneous adipose tissue samples were collected a week before, one day and a week after parturition from cows. Total RNA was extracted from tissue samples quality and quantity of total RNA was analysed using electrophoresis and spectrophotometer. Complementary DNA (cDNA) was prepared from the total RNA for quantitative PCR (qPCR). QPCR was conducted to quantitate expression of the following genes: adiponectin (ADIPOQ), leptin (LEP), peroksisome proliferator activated receptor gamma (PPAR-?), adiponectin receptor-1 (AR1), adiponectin receptor-2 (AR2), lipoprotein lipase (LPL), stearoyl-CoA desaturase (SCD) and hormone-sensitive lipase (HSL). Two genes, AR2 and LEP, were downregulated in group 1. Reduced expression of AR2 in group 1 may relate to an increased insulin resistance. The glucose metabolism was reduced further leading to reduced insulin sensitivity. Lower expression of LEP after parturition indicates usage of energy for milk production. The upregulation of SCD in group 1 before and after calving as well as after calving in group 2 may be a result of the uptake of fatty acids by the mammary tissues. The expression of ADIPOQ, AR1, LPL, PPAR, and HSL did not show any significant changes.

Asteraceae comprises of approximately 10% of all angiosperm plant species. These species are well known for their highly compressed inflorescences known as capitula which consists of morphologically different types of flowers: ray, trans and disc flowers. This immense morphological difference excels Gerbera as an ideal plant to study flower type differentiations. Even though this complex process is governed by several genes, the ray flower identity is believed to be greatly influenced by GhCYC3 promoter mediated gene regulations. In previous studies two TCP transcription factors (TF): GhCIN1and GhCIN2, and two MADS TFs: GAGA1 and RCD5 were identified as the potential upstream regulators of GhCYC3. So, the aim of this study is to test whether these potential upstream regulators physically bind to GhCYC3 promoter in in vitro conditions. In order to achieve the goal, these transcription factor proteins from Gerbera hybrida were successfully expressed in E. coli and purified as fusion proteins to maltose-binding protein (MBP). Physical binding of the purified fusion proteins to the putative target DNA sites in the promoter region of GhCYC3 gene was tested by electrophoretic mobility shift assay (EMSA). The results showed that none of the gerbera transcription factors (GhCIN1, GhCIN2, GAGA1 and RCD5) bind to their putative target sites under the condition tested in this study. However, it might not be justifiable to deduce that these TFs do not interact with GhCYC3 promoter. The absence of in vitro interaction between the tested TFs and GhCYC3 promoter might be caused by either lack of proper folding and activity of the TFs or absence of co-factors which are available in vivo.

The sequencing methods used to study the genome of organisms have become cheaper, resulting in a significant increase in the amount of genomic data available. Knowing the nucleic acid sequence of the DNA does not tell much about an organism. Not without first annotating the genome, which means searching for the locations of genes and defining their products. The programs used for annotation make mistakes and their results must be evaluated in various ways. The vast amount of genomic data encourages fast production of new annotations and this can increase human made errors. Some annotation programs use gene databases, so the number of wrongly annotated genes they contain may increase in the future if the quality control of annotations is not improved. This study examines correlation between selected quality measures and the quality of annotations. The quality metrics used can be divided into two basic types, the first one is based on the basic structures of genes and the second one on comparing the protein product of a gene against a protein database. The study assumes that comparison to a reference is a reliable way to assess the quality of annotations. The comparison is made at genome, exon and nucleotide levels. A single value describing the comparison is calculated at each level. For each gene aligned with a reference gene, sensitivity and specificity are calculated and used to make f-score at the nucleotide level. Four different versions of the wild strawberry (Fragaria vesca) genome and their six annotations were used as data. They were downloaded from the Genome Database for Rosacaea, which is a genome database specializing in rose plants. The correlation coefficients calculated from quality metrics and f-scores were in several cases small but reliable because the p-value was minimal. Correlation coefficients were higher when quality metrics based on protein homology were examined. The correlation coefficient calculated from the mean of the structure-based quality metrics and the f-score received lower values if the studied annotation had a high f-score value. These results detailed in this paper support the view that the selected structure-based quality metrics are not suitable for evaluation of high-grade annotations. They might possibly be used in automated detection of poor-quality annotations. Quality metrics based on protein homology appeared to be promising subjects for further research.

The Finnish dairy cattle population has been subjected to systematic quantitative studies over decades. The Western Finncattle (WFC) has evolved over the last century with a production level comparable to other remaining local breeds in Europe. The heritability is used in designing the data collection and in predicting the changes expected from the selection and the variation parameters are used in constructing the economic selection indices genetic improvement scheme and in computing the bulls’ and cows’ breeding values. WFC has no recent studies on the genetic variation in milk production traits. The thesis research was set to estimate the heritability of milk, fat and protein yield, fat%, protein%, protein-fat ratio and somatic cell count (SCC) and the genetic correlation amongst them. Records from Western Finncattle primiparous cows calving in the period 2002–2016 were used for the genetic analyses. The raw data consisted of 5455 cows distributed across 2512 herds. The variance components were estimated with single and multi-trait animal model using a Bayesian approach and R studio package MCMCglmm. With requiring at least 5 cows in each herd-year subclass in the estimation, the data size was reduced to 1763 cows in 233 herds. The heritability of milk, protein and fat yield, protein%, fat% and SCC was in single (and in brackets for multi) trait analysis 0.36 (0.37), 027(0.30), 0.32 (0.30), 0.61(0.43), 0.52 (0.49) and 0.06 (0.15), respectively. Amongst yield traits and also between the content traits the genetic correlation was high, 0.73–0.94 and 0.43–0.59, respectively. The content traits (with milk yield in the denominator) had a negative genetic correlation with milk yield while no correlation with the protein and fat yield. There was an environmental correlation between content and yield traits for protein and fat. No correlations exist between SCC and other traits except an environmental correlation with milk yield and protein content. Despite the small population size of the WFC population, the effective population size is satisfactory and therefore no reduction in genetic variation is expected. Overall, the analysis on production traits and pedigree data shows that the Western Finncattle have much potential for genetic improvement.

Enterococci is a group of gram positive bacteria part of human intestinal flora. While generally harmless, several species of the group are known to cause severe infections in humans, including bloodstream infections leading to sepsis. Since Enterococci are naturally resistant to many antibiotics, the use of glycopeptides, considered a”last resort” drugs, is common in treatment of enterococcal infections. In recent years, however, the emergence of glycopeptide resistant Enterococci (GRE) has been an increasing concern for clinics and microbiology laboratories around the world, creating a need for fast and accurate screening tests differentiating the glycopeptide resistant Enterococcus strains from the non-resistant ones. In this study, a combined PCR and microarray hybridization based method for identification of the clinically most prevalent GRE was established as a part of commercial sepsis diagnostic test called Prove-it™ Sepsis. Already identifying the most common Enterococcus species (E.faecium and E.faecalis), the detection of glycopeptide resistance causing ligase genes vanA and vanB and species level identification of intrinsically glycopeptide resistant E.gallinarum and E.casseliflavus were added as part of the the test. Primers were designed for sequencing vanA and vanB genes and multiple strains, provided by a Finnish clinical laboratory Huslab, were sequenced. Sequence regions unique to these genes were identified according to sequence alignment data containing the sequenced gene regions and other relevant sequences found in public sequence databases. Based on these data, primers were designed for the amplification of the selected gene regions. For identification of the amplified gene regions, a set of hybridization probes were designed and printed on microarray. In addition, probes for identifying E.casseliflavus and E.gallinarum were designed based on sequence aligment data gathered from Mobidiag Ltd. private biobank. The identification of these species was based on topoisomerase encoding gyrB gene amplified by the Prove-it™ Sepsis broad range PCR. Several primers for the amplification of vanA and vanB genes were designed and one primer pair for each was selected to be integrated to the Prove-it™ Sepsis multiplex-PCR. Similarily, multiple hybridization probes were designed for detecting vanA, vanB, E.casseliflavus and E. gallinarum. Four probes for each target gene region were selected to be integrated to the commercial test. With this modified test, 12 pure culture samples of clinical origin were tested and the results were compared to the ones provided by the laboratory of clinical microbiology of Hôspital de bicêtre (Paris, France). Results provided by the modified PCR and microarray test were identical to the reference results in 11 out of 12 cases.

Great concern is to be addressed to safety measures in order to guarantee work safety when studying novel, possibly pandemic influenza A viruses. These safety measures slow down the research process and their upkeep is expensive. To overcome these hindrances a virus-like-particle (VLP) can be used as a model to replace the need for a live virus. Because VLPs are non-infectious, they are suitable for being used in research where experiments are done with slighter safety precautions. In addition, VLPs are usually highly immunogenic and thus influenza A VLP may function as a model system for further vaccine development. This research was done in a research group, in which a VLP had previously been made with cloning the genes of hemagglutinin (HA), neuraminidase (NA) and matrix 1 (M1) proteins from the year 2009 pandemic Influenza A virus (H1N1)v to a single baculovirus protein expression vector. During this earlier research project it was found problematic that the expression level of a particular gene could not be controlled. In this research, HA, NA and M1 genes were cloned to different baculovirus protein expression vectors so that the expression level of individual genes could be enhanzed with plaque purification and the multiplicity of infection (MOI) adjusted individually for each vector. It was hypothesized that an optimal configuration of MOI rates between vectors could be found in order to maximise VLP production in Spodoptera frugiperda 9 (Sf9) cells. Baculovirus protein expression vectors were made via traditional cloning of the HA, NA and M1 genes into three pAcYM1 baculovirus transfer vectors under polyhedrin promoter, which has been shown to be a strong promoter. Transfer vectors were used to transfer the genes into linearised baculovirus’ genomes by homologous recombination and the genomes were transfected into Sf9 cells to produce recombinant baculoviruses. These expression vectors were plaque-purified and their titers were amplified. Their expression level was studied using SDS-PAGE and coomassie blue analysis and with metabolic labeling using [S35]-labeled methionine. The formation of VLPs was measured with hemagglutination assay when Sf9 cells were co-infected with all three protein expression vectors. It was found that changing expression vectors MOI between 1, 3 and 5 did not have a great impact on protein expression from individual vectors. The presence of NA protein was found to be necessary for the formation of influenza A virus VLPs with a detectable hemagglutination activity. Differences between VLP formations were obtained when MOI rate compositions were changed, but further study is needed to find the significance of this result. The research to find an optimal configuration of MOI rates between vectors is still to be continued.

In addition to being structural components of biological membranes and energy storage of cells, lipids have recently been found to participate as essential players in cell signaling, subcellular transport mechanisms, adjusting functions of integral proteins, and regulation of cell growth and apoptosis. In this study electrospray ionization mass spectrometry (ESI-MS) techniques were used to analyze the phospholipid composition of human bone marrow derived mesenchymal stem cells (BMSC). Numerous chemically distinct lipid species were quantified and the changes in their relative amounts i.e. in the cell’s lipid profile after sequential passaging were followed until senescence (usually from passage 4 up to passage 10, in some cases until p14). Subsequently, the total lipids extracted from the cell pellets were analyzed by triple quadrupole ESI-MS equipment and using lipid-class specific scanning modes. The BMSC lines studied originated from ten donors, five of which were young and five elderly individuals. In culture, the BMSC from both young and aged donors showed time-dependent changes in their phospholipid profiles. The clearest marker findings among individual lipid species were that in phosphatidylcholines (PC) and phosphatidylethanolamines (PE), the species 38:4 (acyl chain pair 18:0/20:4n-6) largely increased towards the late passages, which was seen in the BMSC derived from both the young or aged donors. Thus the reserves of 20:4n-6, the precursor of the eicosanoids having antiproliferative, apoptotic and inflammatory cellular reactions, were increased towards late passages. At phospholipid class level, lysophosphatidylcholine (LysoPC) and phosphatidylinositol (PI) totals, and the ratio of total PI to total phosphatidylserine (PI:PS) were increased from early to latest passages. The results provide new lipid biomarkers to be used for stem cell quality control. The accumulation of polyunsaturated lipid species containing 20:4n-6 or the increase of PI: PS ratio could be potential markers for cell aging and the cells’ poor viability and functionality. The results can be used to develop efficient stem cell therapies and improve patient safety.