Browsing by discipline "Biotechnology"
Now showing items 1-6 of 6
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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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(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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(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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(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.
Now showing items 1-6 of 6