Browsing by discipline "Bioteknik"
Now showing items 1-15 of 15
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(2014)Lymphangiogenesis is the process that leads to the formation of lymphatic vessels from pre-existing vessels. Vascular endothelial growth factor C (VEGF-C), the ma- jor lymphangiogenic growth factor, is produced as an inactive precursor and needs to be proteolytically processed into a mature form in order to activate its receptors VEGFR-3 and VEGFR-2. A deficiency of VEGF-C during embryonic lymphangiogenesis results in embryonic lethality due to the lack of lymphatic vasculature. Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is in a subset of patients associated with mutations in the collagen- and calcium-binding EGF domains 1 (CCBE1 ) gene. CCBE1 and VEGF-C act at the same stage during embryonic lymphangiogenesis and their deficiency results in similar lymphatic defects. The mechanism behind the lymphatic phenotype caused by CCBE1 mutations is un- known. The aim of this study was to investigate the potential link between VEGF-C and CCBE1 that could contribute to the lymphatic phenotype. In this study, 293T cells were used to observe the effect of CCBE1 on VEGF-C pro- cessing. The co-transfection of constructs coding for CCBE1 and VEGF-C showed processing of the inactive pro-VEGF-C into the active, mature form. However, this processing was efficient only in 293T cells. When CCBE1 from 293T supernatant was purified, A disintegrin and metalloproteinase with thrombospondin type 1 motif 3 (ADAMTS3) co-purified with CCBE1. The levels of pro-VEGF-C and active VEGF-C were monitored by immunoblotting or immunoprecipitating metabolically labeled supernatant with specific antibodies or receptors followed by autoradiography. The activity of the processed VEGF-C was verified by proliferation of Ba/F3 cells stably expressing VEGFR-3/EpoR or VEGFR-2/EpoR chimeras. Furthermore, a VEGFR-3 phosphorylation assay was performed in PAE (Porcine Aortic Endotheial) cells to study details of the CCBE1-mediated regulation of VEGF-C. We found that CCBE1 increases the proteolytic processing of pro-VEGF-C, thereby resulting in increased activity of VEGF-C. CCBE1 itself has no effect on VEGF-C activity but regulates VEGF-C by modulating the activity of the ADAMTS3 protease. We also found that both pro- and mature- VEGF-C can bind to VEGFR-3 but only mature form is able to induce VEGFR-3-mediated signaling. In addition to cleaving VEGF-C, ADAMTS3 was found to directly or indirectly mediate CCBE1 cleavage. The N-terminal amino acid sequence of the ADAMTS3-processed VEGF-C confirmed that ADAMTS3 is the protease responsible for the activation of VEGF-C by 293 cells. Hence, we have identified a mechanism that regulates VEGF-C activity. This mechanism suggests the possible use of CCBE1 as a therapeutic means to treat diseases that involve the lymphatic system.
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(2012)Genome wide linkage and association methods are used to map genes affecting traits with genetic predisposition. In this thesis, I compare the methods suitable for quantitative trait mapping in complex, extended pedigrees. As a case study, gene-mapping study of musical aptitude is performed with these methods. Linkage analysis methods are developed for family studies. However, only a few methods are suitable for extended families with a quantitative trait. Three linkage programs were successfully applied for such data in this study. These programs are the SOLAR, JPSGCS and KELVIN. All of these three programs are based on different methods and thus, the same calculations are not repeated. SOLAR is based on the variance components method, JPSGCS on a graphical method and KELVIN on the Bayesian method. Association analysis is also difficult to implement for large pedigrees, because it is best suited for case-control data. Fortunately, methods are extended also for family-based studies. Here, a genomic control method was used to correct for the familial relationships. The method evaluates the relatedness from the whole genome data and the association tests are corrected for the relatedness rates. This method was implemented from the GenAbel program. As a case study, these methods were applied to a musical aptitude study. The musical aptitude is here understood as an ability to perceive the melody, harmony and rhythm of music, and to recognize structures in set of sounds. These abilities were tested with Carl Seashore s tests for pitch and time and Kai Karma's test for auditory structuring. The data consists of 107 pedigrees and 93 sporadic subjects, comprising in total of 915 individuals. Each family includes 2 - 50 individuals. These individuals were genotyped with a SNP chip for over 700,00 SNPs. The linkage analyses revealed several promising loci for the musical aptitude. The best result was located in 4q12 and it was found with all of the three linkage programs. Most of the other results could also be identified with multiple programs, but some differences also occurred. However, none of the findings could be discovered with association analysis, probably due to a too small sample size.
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(2009)Pseudomonas syringae pv. tomato DC3000 is a Gram-negative plant pathogen that causes bacterial speck disease on tomato. The virulence of this bacterium is based on the type III secretion system (T3SS). Similar systems are also used by many other plant and animal pathogens, as well as symbiotic bacteria. The T3SS enables the transfer of specific bacterial virulence proteins from the bacterial cytoplasm into the host cell. This secretion is mediated by a needle-like structure that penetrates the plant cell wall. Once inside the host cells, the effector proteins are capable of shutting down the host’s immune system. However, what happens at the plant cell membrane is not well understood. One of the first bacterial proteins that come into interaction with a host protein during P. syringae pv. tomato DC3000 infection is the HrpZ1 protein that is believed to participate in a membrane interaction, facilitating the transfer of effector proteins. Previous research has shown that HrpZ1 binds to a peptide and using an antiserum raised against this peptide in immunoblotting tests of tomato proteins separated by SDS-PAGE and isoelectric focusing, the target tomato protein of HrpZ1 has been found to be small and acidic. However, this specific protein has not yet been fully characterized. This is why the goal of the work for this thesis was to identify and characterize the target protein of HrpZ1 in tomato. For this purpose, a lambda cDNA expression library from tomato leaves was constructed, followed by immunoscreening of the library with the abovementioned antiserum, and analysing candidate clones by sequencing. Even though a good cDNA library was obtained, the immunoscreenings did not yield satisfactory results. Thus, the pursuit of the HrpZ1 target protein needs to be continued.
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(2017)Trichoderma reesei, an anamorph of Hypocrea jecorina, is a filamentous fungus widely used for producing industrial enzymes. T. reesei is used for both endogenous and heterogenous protein production. The optimization of the production conditions and the effects of extracellular agents to T. reesei s production and secretion capacity are crucial for economically sustainable biotechnical production. The available carbon sources, most commonly different types of sugars, have a significant effect on the production and secretion of enzymes by T. reesei. Genetic modification of the pathways through which the fungi recognizes extracellular signals could bring advancements to industrial enzyme production. Because of T. reesei s potential and use as a production strain, the species is an interesting platform for genetic modifications that would enhance the production capacities. With the current methods the genome editing of T. reesei is however slow, and introducing multiple mutations to a single strain can take years. The aim of this study is to optimize the fairly new CRISPR/Cas9 genome editing system for use in T. reesei. In the CRISPR/Cas9 method, a catalytically active Cas9 enzyme is bound to a specific locus of the genome, guided by a guide RNA and the Watson-Crick base pairing principle. Once in the RNA-guided locus, Cas9 introduces a double stranded break in the DNA, which can be repaired by the cells endogenous non-homologous end joining pathways. This repair is error prone and produces mutations to site of the double stranded break. A donor DNA is often introduced together with the Cas9 and guide RNA. This donor DNA includes sequence homology to the site of interest and allows for the use of the cells homologous repair pathways. In this case, the mutation can be better controlled, and for example the risk of chromosomal mutations is reduced. Currently the CRISPR/Cas9 system is widely used in mammalian cell studies and up to 100% mutation frequencies have been reported in yeast cells. In this study the method is optimized for use in T. reesei. To our best knowledge, the research community has not found an organism in which CRISPR/Cas9 would not function. The question mainly lies on what type of set up and component introduction is suitable for each cell type and research purpose. In this thesis, three putative and one already published genes believed to be involved in hexose sugar sensing will be deleted from a T. reesei production strain with the help of CRISPR/Cas9. The effect of these deletions will be assessed through studying the secretion and activity of endogenous cellulases with enzymatic assays. One sugar transporter that may play a part in glucose sensing was identified in this study. The deletion of this transporter caused a decrease in cellulase production and/or secretion. The three other transporters or sensors did not have a significant effect on cellulase production in spent grain extract and lactose or glucose media. It s possible that these genes are involved in the uptake and use of other carbon sources. The continuous expression of the CRISPR/Cas9 system in T. reesei proved difficult. In the continuous expression method at least one of the CRISPR/Cas9 components, the Cas9 protein or the guide RNA, is produced in the cells in vivo. Neither was achieved in this study. Instead, a fully synthetic method in which the Cas9 is transformed into the cells as a protein along with an in vitro produced guide RNA was set up and produced up to 1000 × higher mutation frequencies when compared to the traditional transformation method used for T. reesei. This study also demonstrates a simultaneous deletion of two genes in T. reesei. To the best of our knowledge, multiple simultaneous gene modifications have never been achieved in T. reesei.
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(2015)Studies of population structure are motivated by the need to understand population history and to have well-characterised groups of individuals in studies of genetics of diseases and traits. A standard method to analyse genetic population structure is principal component analysis (PCA). A disadvantage of PCA is that it can reliably handle only independent genetic markers. This means that the genetic markers that are correlated with other genetic markers have to be excluded from the data. This leads to a loss of information. In 2012, Lawson et al. published a chromosome painting method that can utilise haplotype information, i.e. information from correlated markers, and thus it can detect more subtle differences in populations than the standard PCA. This thesis studies two questions. The first question is whether the chromosome painting method can provide more precise genetic clustering of geographically defined Finnish groups than the standard PCA method. The second question is whether the chromosome painting method can reveal new details of population structure in Finland. The data used in this study are from the FINRISK Study survey of 1997. This cohort includes the genotype data of about 4,000 individuals and the information about individuals and their parents birthplaces. 345 Individuals were randomly chosen from the cohort in such a way that both of their parents were originated from the same province. Ten provinces of Finland were used as study groups for the method comparison. First, the data were analysed with SmartPCA (a standard PCA method) and ChromoPainter (the chromosome painting method) and the results were compared both visually and quantitatively. Finally, the individuals were assigned to populations based on the ChromoPainter result using FineSTRUCTURE program and these genetic populations were compared to the geographic origin of the individuals. The results showed that the chromosome painting method clustered seven out of ten groups significantly tighter than the standard PCA. Nevertheless, SmartPCA was faster and easier to use than ChromoPainter. The main population genetic division was found between the eastern and western parts of Finland, which was consistent with earlier studies. All in all, 15 populations were detected and the results revealed that they were geographically clustered. The genetic populations correlated well with the borders of Finnish provinces and counties. As the first conclusion, the chromosome painting method was able to give more precise results than the standard PCA but the standard PCA is still more suitable for quick preliminary analyses of genetic data. As the second conclusion, the chromosome painting method was able to detect detailed subpopulation structure in Finland and these populations are geographically clustered. Results provide an excellent basis for the future studies of population structure and genetic diseases in Finland.
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(2013)The next-generation sequencing (NGS) platforms create a large amount of sequence in short amount of time, when compared to first generation sequencers. An overview of the NGS platforms is provided with more in-depth look into Illumina Genome Analyzer II as that is used to create the data for the thesis. There were two main aims in this thesis. First, to create a pipeline which can be used to analyse genomic sequencing. Second, to use the pipeline to compare whole human exome capture methods from two manufacturers, Roche Nimblegen and Agilent. The pipeline is describe in detail in material and methods. All the inputs for the pipeline are described and examples shown. In the pipeline the given sequences are first aligned against the reference genome. Then various separate analysis is performed to retrieve variants and coverage of the sequencing. Supplementary results include paired-end anomalies, larger insertion and deletion polymorphisms and assembly of non-aligned sequences. The two capture methods are also described and changes to the manufacturers' recommended protocols are listed. Finally, the section has the options and various inputs used in the pipeline runs of the exome data. The results of the pipeline is a basic level of analysis of the sequencing as well as various graphs showing the quality of the run. All the output files intended for user are described. By using the results of the pipeline, the user can do more in-depth analysis as required by the project. When comparing the two exome capture methods, the Nimblegen capture was shown to be more efficient in capturing the CCDS exome. While the Agilent capture kit provided better one fold coverage over the exome, higher fold coverage (over 10 fold), which is required for reliable variant calling in nextgeneration sequencing, was better reached using the Nimblegen capture kit. Also, significantly fewer false positive paired-end anomalies were observed in the library created by using the Nimblegen capture.
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(2017)Lactobacilli are gram-positive lactic acid bacteria with wide beneficial properties for human health and food production. Today most of the fermented products and probiotic foods are produced by lactobacilli species. One of the most using area of lactobacilli species is fermented products especially dairy products. Lactobacilli species can be used as starter or adjunct cultures in dairy products and play important role for preservation and quality, texture and flavor formation. Additionally, probiotic properties of lactobacilli species provide several health effect for human by stimulation of immune system and protection against pathogens. Lactobacillus rhamnosus LC705 is a facultatively heterofermentative type lactobacilli which is used in production of dairy products as adjunct starter and protective culture. The complete and annotated genome sequence of L. rhamnosus strain LC705 published on 2009. Known characteristics of L. rhamnosus strain LC705 are food preservation, toxin removal and health benefits when combined with other probiotic strains. However, molecular mechanism behind these characteristics are not known or not clearly understood. To get further insight on these properties and roles in cheese ripening of strain LC705, we re-annotated genome of the LC705 with updated methods and databases, analyzed metabolic pathways of LC705, and performed RNA-seq experiment to determine gene expression changes of LC705 during warm room (25 °C) and cold room (5 °C) cheese ripening process. Several un-characterized proteins of LC705 were annotated (77) and 1197 enzyme commission (EC) numbers are added to annotation file with re-annotation of genes of LC705. More importantly, re-annotation provided us 72 new pathways of LC705 which is 35% of the entire collection of 201 pathways. Analyzes of pathways showed that genome of LC705 has responsible genes for production of flavor compounds such as acetoin and diacetyl which are provide buttery flavor to dairy products, and hydrogen sulfide which is a volatile sulfur compound that cause unlikeable odor. Additionally to flavor compounds, we defined genes that produce anti-fungus compounds and bacteriocin which provide food preservation characteristic to LC705. Determination of gene expression respond of LC705 during warm room and cold room cheese ripening process with RNA-Seq showed that central metabolism genes that responsible for lyase activity, degradation activity, disaccharides and monosaccharides metabolism are warm induced genes. The genes play role in citrate metabolism pathways were significantly down-regulated during cold room, citrate degradation pathways are critical for buttery flavor products, therefore buttery flavor compounds are produced by LC705 during warm room. Finally, during cold room ripening, the genes of LC705 that produces ethanol and acetyl-CoA from pyruvate was up-regulated, so we may say that LC705 uses pyruvate to produce ethanol and acetyl-CoA instead of lactic acid.
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Identification of new protein-protein interactions with NUAK2 by proximity-dependent biotin labeling (2017)In this Master’s project, I have studied a mammalian serine-threonine kinase NUAK2 implicated in human disease but whose molecular functions and interacting proteins are as of yet poorly characterized. The goal was to identify new interacting proteins to increase understanding of the molecular functions and potentially link to human physiology and disease. Recent work from the host lab shows NUAK2 loss in cultured primary cells mimics loss of the tumor suppressor LKB1 which also acts upstream of NUAK2, together suggesting NUAK2 could be involved in tumor suppression. Currently, only two protein-protein interacting proteins with NUAK2 have been identified: NUAK2 is targeted to actin stress fibers by the myosin phosphatase Rho-interacting protein (MRIP), and it is involved in regulating cell contractility by affecting indirectly the phosphorylation cycle of the myosin light chain through inactivation of the myosin phosphatase target subunit 1 (MYPT1). In this project, I utilized a novel protein-protein interaction screening method that utilizes proximity-dependent biotin labeling to identify new interacting proteins with NUAK2 in human embryonic kidney cells (HEK 293). This method is based on fusing an E.Coli promiscuous biotin ligase, BirA*(R118G), to the investigated protein. The BirA*(R118G) ligase biotinylates all the proteins in close proximity of the fusion protein creating a history of protein-protein associations over time. Afterwards, the biotinylated proteins can be isolated by affinity purification methods and identified by mass-spectrometry. The screening identified the previously known interaction partners of NUAK2 indicating it was technically successful. In addition, I also identified in total 108 novel potential protein interaction partners for NUAK2. One of the top hits was Cytospin-A, a cross-linking protein between microtubules and actin cytoskeleton, supporting a role of NUAK2 as regulator of cytoskeleton. Supporting the validity of our finding, Cytospin-A depletion in mammalian cells causes defective actin-cytoskeleton reorganization, a very similar phenotype seen with NUAK2 depletion. In future studies, I will continue to investigate the specific role of NUAK2 and Cytospin-A aiming for detailed information on the function of NUAK2 in regulation of microtubules and actin cytoskeleton. Validation of some of the other identified interactions is expected to provide novel insights to the biology and role of NUAK2 in LKB1 tumor suppressor functions.
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(2013)Lipoproteins play a central role in the disease mechanisms of cardiovascular diseases (CVD) and therefore they have been studied widely. They carry several classes of apolipoproteins where apo-A1 and apo-B are the major classes. The sucrose based sequential lipoprotein isolation method can retrieve the lipoprotein fractions suitable for lipidomics analyses. The main lipoprotein classes are very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high density lipoprotein (HDL) that can be isolated easily by their density from human blood plasma or serum. Lipidomics analyses can quantify lipids that lipoproteins carry in the circulation. Mainly they carry cholesterol and its esterified forms, glycerolipids, small amounts of sphingolipids and phospholipids form their monolayer membrane. The isolation method was set-up together with scaled-down sample volumes. The protein and lipid content of the main lipoprotein fractions were evaluated by electrophoresis analysis, various enzymatic assays and lipidomics analyses. The total protein and apolipoprotein content was found to be similar as in the literature. Apo-B was found to be the main apolipoprotein in the VLDL and the LDL fractions whereas apo-A1 was the main apolipoprotein in the HDL fractions. Triglycerides (TG) were measured by enzymatic analysis and TG was mainly found in LDL and VLDL. The lipidomics analyses demonstrated the lipid content of the lipoproteins were similar as in the literature with minor changes. The main lipid class found in all the lipoproteins was cholesteryl esters (CE) followed by phosphatidylcholines (PC) that are commonly found in cell membranes. Sphingolipids such as ceramides were also detected in lipid class level only in small quantities in the lipoprotein fractions. The low initial sample volume did not correlate linearly with higher sample volume and low sample volume is not recommended to use in this specific isolation method. Based on the results of the comprehensive screening of isolated lipoproteins the isolation method was successfully established.
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(2011)Ewing sarcoma is an aggressive and poorly differentiated malignancy of bone and soft tissue. It primarily affects children, adolescents, and young adults, with a slight male predominance. It is characterized by a translocation between chromosomes 11 and 22 resulting in the EWSR1-FLI1fusion transcription factor. The aim of this study is to identify putative Ewing sarcoma target genes through an integrative analysis of three microarray data sets. Array comparative genomic hybridization is used to measure changes in DNA copy number, and analyzed to detect common chromosomal aberrations. mRNA and miRNA microarrays are used to measure expression of protein-coding and miRNA genes, and these results integrated with the copy number data. Chromosomal aberrations typically contain also bystanders in addition to the driving tumor suppressor and oncogenes, and integration with expression helps to identify the true targets. Correlation between expression of miRNAs and their predicted target mRNAs is also evaluated to assess the results of post-transcriptional miRNA regulation on mRNA levels. The highest frequencies of copy number gains were identified in chromosome 8, 1q, and X. Losses were most frequent in 9p21.3, which also showed an enrichment of copy number breakpoints relative to the rest of the genome. Copy number losses in 9p21.3 were found have a statistically significant effect on the expression of MTAP, but not on CDKN2A, which is a known tumor-suppressor in the same locus. MTAP was also down-regulated in the Ewing sarcoma cell lines compared to mesenchymal stem cells. Genes exhibiting elevated expression in association with copy number gains and up-regulation compared to the reference samples included DCAF7, ENO2, MTCP1, andSTK40. Differentially expressed miRNAs were detected by comparing Ewing sarcoma cell lines against mesenchymal stem cells. 21 up-regulated and 32 down-regulated miRNAs were identified, includingmiR-145, which has been previously linked to Ewing sarcoma. The EWSR1-FLI1 fusion gene represses miR-145, which in turn targets FLI1 forming a mutually repressive feedback loop. In addition higher expression linked to copy number gains and compared to mesenchymal stem cells, STK40 was also found to be a target of four different miRNAs that were all down-regulated in Ewing sarcoma cell lines compared to the reference samples. SLCO5A1 was identified as the only up-regulated gene within a frequently gained region in chromosome 8. This region was gained in over 90 % of the cell lines, and also with a higher frequency than the neighboring regions. In addition, SLCO5A1 was found to be a target of three miRNAs that were down-regulated compared to the mesenchymal stem cells.
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(2009)Environmental concerns and limited availability of fossil hydrocarbons have boosted the research of renewable feedstocks and their processing into fuels and chemicals. Currently, vast majority of transportation fuels and bulk chemicals are refined from crude oil, but renewable lignocellulosic plant biomass has long been recognised as potential feedstock for liquid fuel and chemical production. Several alternative processes exist for biomass refining, lignocellulose-to-ethanol process being among the most studied processes. First, lignocellulose is pretreated in order to deconstruct the recalcitrant structures of plant cell walls and expose cellulosic fibrils. Subsequently, biotechnical process utilises cellulolytic enzymes of fungal origin to depolymerise cellulose down to glucose monomers and oligomers. Monomeric sugars serve as a source for platform chemicals in further conversions. Lignocellulose consists mainly of cellulose, hemicellulose and lignin. It is generally accepted that lignin has an inhibitory effect during enzymatic hydrolysis of cellulose and part of this effect is caused by irreversible cellulase adsorption on lignin. Fungal cellulase system consists of several enzyme components that contribute to the effective degradation of insoluble cellulosic substrate. Cellulases are traditionally divided to three groups according to enzymatic activity: exoglucanases, endoglucanases and ?-glucosidases. Different enzyme components are shown to have different affinity to lignin which enables screening or engineering of weak lignin-binding enzymes. However, too little is still known about enzyme-lignin interactions and competitive nature of enzyme binding on lignin. In this study, lignin-rich residues were isolated from steam pretreated spruce (SPS) using three different methods: enzymatic hydrolysis, acid hydrolysis and alkali extraction. Lignin residues were characterized and used in adsorption studies with commercial cellulase preparations from Trichoderma reesei (Celluclast 1.5L) and Aspergillus niger (Novozym 188). Enzyme activity measurements and protein analytics were employed to reveal competitive adsorption of cellulases and catalytic activity of solid-bound enzymes. Results showed that T. reesei enzymes had high affinity on lignocellulosic SPS and all SPS-derived lignins, but enzyme activity measurements revealed considerably divergent competitive adsorption patterns. Among all the isolated lignins, lignin-rich residue obtained by enzymatic hydrolysis of SPS and subsequent protease purification was evaluated as most suited adsorption substrate for further adsorption studies and screening purposes. ?-glucosidases from T. reesei and A. niger were shown to have highly distinctive adsorption behaviour on the lignin-rich substrates: A. niger ?-glucosidase lacked affinity to lignin, whereas T. reesei ?-glucosidase adsorbed to all lignin-rich particles. Lignin-bound Trichoderma reesei endoglucanases and CBH I exoglucanase were shown to retained high activity towards soluble substrates used in activity measurements. On the contrary, same enzymes were unable to processively hydrolyze insoluble crystalline cellulose.
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(2011)Chronic myeloid leukemia (CML) is one of the most studied human malignancies. It is caused by an autonomously active tyrosine kinase BCR-ABL, which is a result from a translocation between chromosomes 9 and 22 in the hematopoietic stem cell. As an outcome, a Philadelphia (Ph) chromosome is formed. BCR-ABL causes disturbed cell proliferation among other things. Although targeted tyrosine kinase inhibitor therapy has been developed in the beginning of the millenium and the survival rate has increased significantly, it is still not known why some patients benefit more from the treatment than others. Furthermore, the therapy is not considered to be curative. Before the era of tyrosine kinase inhibitors, the first-line treatment for CML was interferon-? (IFN-?). However, only a small proportion of patients benefitted from the treatment. Of these patients, a few were able to discontinue the treatment without renewal of the disease. The mechanism of IFN-? is not completely understood, but it is believed that differences in the immune system can be one of the reasons why some patients have better therapy response. Kreutzman, Rohon et al. have recently discovered that patients who have been able to stop IFN-? treatment have an increased number of NK- and T-cells. They also have a unique clonal T-cell population and more cytotoxic CD8+ T-cells and less CD4+ T-cells. The aim of this master’s thesis was to study the function of T- and NK-cells in IFN-? treated patients. Although it was shown earlier that IFN-? treated patients have increased NK-cell count, the function of these cells was unknown. Therefore, we have now investigated the killing potential of patients’ NK-cells, their activation status and cell surface antigen expression. In addition, we have also studied the activation status of patients’ T-cells and their cytotoxic properties. We observed that NK-cells from patients treated with IFN-? are unable to kill leukemic cells (K562) than NK-cells from healthy controls. In addition, patients on IFN-? treatment have more active T-cells and their NK-cells have an undifferentiated immunoregulatory phenotype. Patients that have been able to stop the treatment have anergic T-and NK-cells. As a conclusion our results suggest that IFN-? therapy induces increased NK-cell count, NK-cell immunoregulatory functions and more active T-cells. After stopping IFN-? therapy, NK- and T-cells from CML patients restore anergy typical for CML.
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Optimization of sample preparation for next-generation sequencing with Illumina Genome Analyzer II (2010)The growing interest for sequencing with higher throughput in the last decade has led to the development of new sequencing applications. This thesis concentrates on optimizing DNA library preparation for Illumina Genome Analyzer II sequencer. The library preparation steps that were optimized include fragmentation, PCR purification and quantification. DNA fragmentation was performed with focused sonication in different concentrations and durations. Two column based PCR purification method, gel matrix method and magnetic bead based method were compared. Quantitative PCR and gel electrophoresis in a chip were compared for DNA quantification. The magnetic bead purification was found to be the most efficient and flexible purification method. The fragmentation protocol was changed to produce longer fragments to be compatible with longer sequencing reads. Quantitative PCR correlates better with the cluster number and should thus be considered to be the default quantification method for sequencing. As a result of this study more data have been acquired from sequencing with lower costs and troubleshooting has become easier as qualification steps have been added to the protocol. New sequencing instruments and applications will create a demand for further optimizations in future.
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(2010)Actin stress fibers are dynamic structures in the cytoskeleton, which respond to mechanical stimuli and affect cell motility, adhesion and invasion of cancer cells. In nonmuscle cells, stress fibers have been subcategorized to three distinct stress fiber types: dorsal and ventral stress fibers and transverse arcs. These stress fibers are dissimilar in their subcellular localization, connection to substratum as well as in their dynamics and assembly mechanisms. Still uncharacterized is how they differ in their function and molecular composition. Here, I have studied involvement of nonmuscle alpha-actinin-1 and -4 in regulating distinct stress fibers as well as their localization and function in human U2OS osteosarcoma cells. Except for the correlation of upregulation of alpha-actinin-4 in invasive cancer types very little is known about whether these two actinins are redundant or have specific roles. The availability of highly specific alpha-actinin-1 antibody generated in the lab, revealed localization of alpha-actinin-1 along all three categories of stress fibers while alphaactinin-4 was detected at cell edge, distal ends of stress fibers as well as perinuclear regions. Strikingly, by utilizing RNAi-mediated gene silencing of alpha-actinin-1 resulted in specific loss of dorsal stress fibers and relocalization of alpha-actinin-4 to remaining transverse arcs and ventral stress fibers. Unexpectedly, aberrant migration was not detected in cells lacking alpha-actinin-1 even though focal adhesions were significantly smaller and fewer. Whereas, silencing of alpha-actinin-4 noticeably affected overall cell migration. In summary, as part of my master thesis study I have been able to demonstrate distinct localization and functional patterns for both alpha-actinin-1 and -4. I have identified alpha-actinin-1 to be a selective dorsal stress fiber crosslinking protein as well as to be required for focal adhesion maturation, while alpha-actinin-4 was demonstrated to be fundamental for cell migration.
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(2008)Anabaena is a common member of the phytoplankton in lakes, reservoirs and ponds throughout the world. This is a filamentous, nitrogen-fixing cyanobacterial genus and is frequently present in the lakes of Finland. Anabaena sp. strain 90 was isolated from Lake Vesijärvi and produces microcystins, anabaenopeptilides and anabaenopeptins. A whole genome shotgun sequencing project was undertaken to obtain the complete genome of this organism in order to better understand the physiology and environmental impact of toxic cyanobacteria. This work describes the genome assembly and finishing, the genome structure, and the results of intensive computational analysis of the Anabaena sp. strain 90 genome. Altogether 119,316 sequence reads were generated from 3 genomic libraries with 2, 6 and 40 kb inserts from high throughput Sanger sequencing. The software package Phred/Phrap/Consed was used for whole genome assembly and finishing. A combinatorial PCR method was used to establish relationships between remaining contigs after thorough scaffolding and gap-filling. The final assembly results show that there is a single 4.3 Mb circular chromosome and 4 circular plasmids with sizes of 820, 80, 56 and 20 kb respectively. Together, these 4 plasmids comprise nearly one-fifth of the total genome. Genomic variations in the form of 79 single nucleotide polymorphisms and 3 sequence indels were identified from the assembly results. Sequence analysis revealed that 7.5 percent of the Anabaena sp. strain 90 genome consists of repetitive DNA elements. The genome sequence of Anabaena sp. strain 90 provides a more solid basis for further studies of bioactive compound production, photosynthesis, nitrogen fixation and akinete formation in cyanobacteria.
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