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Diagnostics of Hypercoagulation Status by D-dimer Concentration Measurement
STIEBLEROVÁ, Romana
In common thrombofilia is the state of the vessel system in arterial, venous or microcirculation part when the probability of thrombs-creation is increased, but thrombs are not present yet. It is the pre-state of thrombotization. Thrombofilia is denoted as hypercoagulation status. Thrombofilia also is a common term for hereditary and acquired prothrombotic states in arterial, venous and microcirculation system. Progress in thrombotic and anticoagulation treatment and high-powered clinical research in pathogenesis together enable the radical progress in modern conception of pathophysiology in clinical diagnostic and introduce usage of very efficient treatments. Results of big multi-central studies show clearly how the precise fibrinolytic and coagulation tests and treatments can help in long-term surviving of patients with thrombosis. The search part of this study shows a summary of last information in pathophysiology and last potentialities in laboratory tests and interpretations focused on clinical exposure and diagnostic of PE and DVT. Inspiration for this study are results published in JOURNAL OF THROMBOSIS AND HAEMOSTASIS 10 (7): Penaloza, A., Roy, P. -M., Kline, J., Verschuren, F., Le Gal, G., Quentin-Georget, S. , Delvau, N. , Thys, F. Performance of age-adjusted D-dimer cut-off to rule out pulmonary embolism. 1291-1296, 2012. doi:10.1111/j.1538-7836.2012.04769.x and HAEMATOLOGICA-THE HEMATOLOGY JOURNAL 97(10) Douma, Renee A.; Tan, Melanie; Schutgens, Roger E. G.; et al. Using an age-dependent D-dimer cut-off value increases the number of older patients in whom deep vein thrombosis can be safely excluded. 1507-1513, 201, doi:10.3324/haematol.2011.060657. In this part also the need of Good Laboratory Praxis for pre-analytics extraction and transportation of whole blood samples for coagulation and fibrinolytic tests is highlighted. The practical part of this study covers either the laboratory results aimed to confirm the diagnosis or the results of D-dimer concentration done for preventive reasons and selected in specific files. D dimer is the specific fissile product of fibrin and the presence of D-dimer in plasma testifies the activation of coagulation and fibrinolysis. D-dimer tests were done on the automatic coagulation analyzer ACT Elite Pro. D-dimer test has been done by immunological method based on reaction between antigen and antibody. Analyzer ACL Elite PRO uses nephelometry for detection of blood clogs and reads the intensity of scattered 90° angle light in the sample. Results of sonography are from data stored in hospital information system FONS Akord STAPRO Hospital Jindřichův Hradec a.s. All data were collected in several data-files. This study verificates the hypothesis of predicted difference of D-dimers in patients from preventive file and patients in file with thrombofilia diagnosis. The hypothesis of D-dimer test positivity in venous thrombosis was confirmed as well, D-dimer as a marker of thrombofilia shows an actual activation in-vivo system and increased D-dimer concentration confirmed venous thrombosis DVT together with a positive sonography. The most interesting and the most important result of this study is the confirmation of the hypothesis of using an age-dependent D-dimer cut-off in patients over 50 of age with suspect DVT or/and PE diagnosis. The importance of using an age-dependent D-dimer cut-off was published in articles in JOURNAL OF THROMBOSIS AND HAEMOSTASIS 10 (7): Penaloza, A., Roy, P. -M., Kline, J., Verschuren, F., Le Gal, G., Quentin-Georget, S. , Delvau, N. , Thys, F. Performance of age-adjusted D-dimer cut-off to rule out pulmonary embolism. 1291-1296, 2012. doi:10.1111/j.1538-7836.2012.04769.x and HAEMATOLOGICA-THE HEMATOLOGY JOURNAL 97(10) Douma, Renee A.; Tan, Melanie; Schutgens, Roger E. G.; et al. Using an age-dependent D-dimer cut-off value increases the number of older patients in whom deep vein thrombosis can be safely excluded.
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Value of the Antibodies Testing in the Diagnostics of Tick Borne Encephalitis
CHADTOVÁ, Marie
The Diagnostic Value of the Antibody Level Testing in Tick Born Encephalitis. This bachelor thesis is focused on the antibody testing and its diagnostic value in Tick Born Encephalitis.Data for this thesis were obtained at the laboratory of Virology, Dpt. of Medical Microbiology of the Cenral Laboratories, Hospital České Budějovice. The data were collected between January 1st and December 31st 2014. The thesis is divided in two parts: the theoretical and the experimental part. The theoretical part covers the history of the disease, its geographic distribution and the ways of the transmission of the virus. The disease was first described by Austrian physician Hans Schneider in 1931 as a periodically occuring disease. He called it "Epidemische acute Meningitis serosa". The virus was first described as the cause of the disease in Russia in 1937. In Czechoslovakia, the virus was described in 1947 in Prague by virologists František Galia and Josef Rampas. The first succesful virus isolation in Czechoslovakia was from Beroun district, the next successful isolation was in Vyškov district made by Dr. Krejčí. The virus was later isolated in other European countries as well. The hypothesis, that the virus is transmitted by ticks, was proved by Dr. Rampas and Dr. Galia by the isolation of the virus directly from the ticks Ixodes ricinus, collected in the Beroun district. The transmission of the virus by alimentary route was first described in 1951 near east Slovakian town Rožňava. The virus was transmitted by drinking nonpasteurized sheep milk. The laboratory infection is possible, when laboratory staff works with the viable virus. The Tick Borne Encephalitis virus is part of the genus Flavivirus , family Flaviviridae. It is an enveloped virus with cubic symetry. Virus particles are round shaped, with diameter 5060 nm. The central part is nucleus, containing viral RNA, enveloped by protein capsid and lipoprotein envelope. Lipoprotein envelope is double lined and contains important antigens - glycoprotein E and menbrane protein M. Tick Borne Encephalitis is transmitted by ticks. The incidance is therefore closely connected to the activity of the ticks in nature. The activity of the ticks depends on the season, temperature, humidity and altitude. The presence of people in focal point is also essential. Tick Borne Encephalitis is an infection with the natural focal point. Its natural reservoir are mainly small rodents and other wild animals. Human activity in nature can lead to the transmission of the disese to humans. Human are a blind end of the transmission chain, no interhuman transmisson was described. The virus penetrates the organism via the tick bite. The virus penetrates to the skin from salivary glands of the tick through the small wound. The primary multiplication starts there. The virus is later spread by lymphatic system to the blood stream. This corresponds with the first phase of the infection. The second phase of the infection is accompanied by secondary viremia, the virus multiplies in various tissues and spreads into the central nervous system. The incubation period before the beginning of the first phase is 714 days. The first phase may proceed under the clinical picture of mild, flu- like virosis with the increased temperature, muscle pain and fatigue. This period lasts 1 to 2 days, maximum 6 days. After the first phase there is a period of approximately one week without any signs of the disease. After this period the second phase may occur, with sthe signs of meningitis, menigoencephalitis or myelitis. The treatment is symptomatic. To diminish the symptoms of the disease, analgetics, antipyretics , anticonvulsives and sometimes antiedematic treatment is indicated. The epidemiological mesures are based on education and vaccination. The vaccine is available since the eigties of the last century. Laboratory diagnostic may use direct or indirect detection of the virus. The methods of direct detect
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Studies on NKR-P1A and NKR-P1B receptors expressed in eukaryotic organisms
Ivanova, Lyubina ; Rychlovský, Petr (referee) ; Bezouška, Karel (advisor)
NK (natural killer) cells, with their ability to identify antigens and extraneous substances, available in the organism through various moleculary receptors, are an important component of the immune system. The NKR-P1A and NKR-P1B proteins belong to the lectin receptors of natural killer cells. Primary ligands of lectin receptors comprise terminal oligosaccharides of glycoproteins on the surface of target (e.g. tumor) cells. The interaction between carbohydrate structures on the surface of antigens and their binding partners on NK receptors is followed by triggering the effector function of NK cells against the targets. The NK cells and NK receptors findings and their interactions with ligands are greatly utilized in the treatment of cancer, viral and autoimmune diseases. Heterologous protein production in the eukaryotic organism brings a lot of advantages. Unlike the prokaryotic organism, the methylotrophic yeast Pichia pastoris has the capability of performing many posttranslational modifications resulting in production of biological active protein molecule. Usually, the P. pastoris expression system disposes of high level protein expression and is also generally regarded as being faster, easier, and less expensive to use than expression systems derived from other eukaryotes. In this thesis, I...
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Functional role of purinergic P2X receptors in the supraoptic nuclei of the rat and structure-function relations of recombinant P2X receptors.
Vávra, Vojtěch ; Zemková, Hana (advisor) ; Novák, František (referee) ; Vyklický, Ladislav (referee)
Purinergic P2X receptors are non-selective cationic channels gated by extracellular ATP. Up to now, seven mammalian subunits, termed P2X1-X7, have been cloned and characterized. These receptors comprise a new membrane channel family with distinct structural and functional features. P2X receptors take part in a signalling network called "purinergic signalling" which is widely exploited in both somatic and neuronal tissues. In the central nervous system, they are highly expressed in the hypothalamus and hypophysis, where they participate in the regulation of homeostatic and reproductional functions. The main focus of my Thesis is on the expression and functional role of P2X receptors in supraoptic nuclei of the rat hypothalamus. These nuclei contain two populations of magnocellular neurons which produce either oxytocin or arginine-vasopressin. Delivery of the hormones into the systemic blood relies on the electrical activity of supraoptic neurons, which is in turn governed by the incomming synaptic inputs. It has been recently shown, that the process of hormone release from supraoptic neurons is regulated by extracellular ATP. However, purinergic signals that regulate hormone secretion are not well understood. The aim of my study was to identify subtypes of P2X receptors expressed in the supraoptic...
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Raman optical activity and conformation of structurally important groups in peptides and proteins
Kubáňová, Markéta ; Baumruk, Vladimír (advisor) ; Kapitán, Josef (referee)
Raman optical activity (ROA) represents a modern spectroscopic technique that can be applied to a wide range of chiral molecules starting from small organic molecules up to complex biomolecular systems. Among other things ROA provides information about solution structure of peptides and proteins. The aim of the thesis was to determine the relationship between three-dimensional structure and Raman optical activity of disulphide and amide groups in peptides. Characteristic band patterns of the polyproline II conformation (left-handed 31-helix) were found in the ROA spectra of poly(Pro-Gly-Pro), oxytocin and hinge peptide linked to the antigen sequence. ROA signal in the S-S and C-S stretching region was observed in ROA spectra of model cyclodextrin compounds connected with disulphide bonds. Positive ROA band in the S-S stretching region was found in the ROA spectrum of oxytocin (the peptide with one S-S bridge). According to the theoretical studies of model disulphides, positive ROA signal in this region indicates positive dihedral angle C- S-S-C. This result is in agreement with the crystal structure. We have also worked on extension of ROA measurements to the hydrogen stretching region (2500-3200 cm-1).
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NKR-P1C receptor-carbohydrate interaction contributes to antitumor immune response via activation of NK and B cells
Hulíková, Katarína ; Fišerová, Anna (advisor) ; Kročová, Zuzana (referee) ; Turánek, Jaroslav (referee)
Lectin - saccharide interactions and the involved receptors are currently intensively studied for their important role in antimicrobial as well as antitumor immunity. The major cell types participating in carbohydrate recognition are NK, NKT, and B cells. The differentiation of B lymphocytes could be induced by activated NK cells via direct intercellular contact and/or IFN-γ release. Our research is focused on NK cell receptors of C-type lectin-like family recognizing carbohydrate epitopes of glycoproteins. Synthetic glycoconjugates with terminal N- acetyl-D-glucosamines on polyamidoamine (GN8P) or calix[4]arene (GN4C) scaffold used in this study, exerted the highest binding affinity to activating isoforms of rodent NKR-P1 (A and C) receptor. The aim of the presented dissertation thesis was to elucidate, how Nkr-p1c gene divergence, between C57BL/6 (NK1.1-positive, NKR- P1CB6) and BALB/c (NK1.1-negative, NKR-P1CBALB/c) mouse strains, could affect NK cell activation and subsequent triggering of B lymphocyte effector functions using GN8P and GN4C as NKR-P1C prototype ligands. We demonstrated, that GN8P increased mRNA expression for NKR-P1C receptor, IFN-γ synthesis and lytic activity of NK cells, antigen-specific (anti-KLH, anti-DNP, and anti-B16F10) IgG (particularly IgG2a) formation, number of...
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Construction of various types of vaccines based on the structural proteins of mouse polyomavirus and analysis of immune response after their administration to mice
Hrušková, Veronika ; Forstová, Jitka (advisor) ; Roubalová, Kateřina (referee) ; Reiniš, Milan (referee)
Conclusion Humoral and cellular immune responses developed in mice after intranasal delivery of model mouse polyomavirus derived VLPs carrying epitope of enhanced green fluorescence protein (EGFP) Model chimeric EGFP-VLPs were purified and used for immunization of mice. Immune response of immunized animals was examined. No specific antibodies against EGFP protein, but high titers of specific antibodies against major structural protein VP1 were developed in the sera of immunized animals. Splenocytes derived from immunized animals secreted IL-2 and IFN-γ after their antigen (EGFP or VP1) restimulation. Proliferation of CD4+, but not CD8+ T cells from immunized mice after the stimulation with both EGFP and VP1 was observed. No EGFP specific cytotoxic activity of splenocytes from immunized mice was detected. The presentation of EGFP-VLPs in the context ofMHC class II was blocked by inhibitors of endo-lysosomal as well as proteasomal compartments. Changes in the numbers of CD25+Foxp3+ subpopulation of CD4+ T cells were observed in the spleens if immunized mice. Chimeric VLPs derived from mouse polyomavirus carrying epitopes of human Bcr-Abl fusion protein (Bcr-Abl VLPs) Chimeric Bcr-Abl VLPs carrying 171 amino acids sequence of Bcr-Abl protein (containing Bcr-Abl breakpoint region) were prepared. Chimeric VLPs...
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Cloning, expression and purification of human AKR1C4
Kosánová, Kateřina ; Wsól, Vladimír (advisor) ; Novotná, Eva (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Kateřina Kosánová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1C4. AKR1C4 is one of four enzymes in men belonging to subfamily of aldo-keto reductases AKR1C. It is monomeric cytosolic protein with length of 323 amino acids expressed in liver. It plays an important role both in metabolism of endogenous and exogenous substances. It is involved in the metabolism of steroid hormones and bile acids and many drugs, for example tibolone and naltrexone. It also plays a role in activation of some cancerogenic substances, e.g. PAHs. cDNA of enzyme was delivered in cells of E. coli DH10B, in pDNR-LIB vector. After lysis of cells and isolation of plasmid, the coding sequence was amplified by PCR. Afterwards it was ligated into vector pET-28b, thanks to added restriction sites for Nde I and Xho I endonucleases in designed PCR primers. The recombinant plasmid prepared by this way was transformed by heat shock to cells E. coli HB101. After amplification of ligated plasmid it was transformed to E. coli BL21. Adjusted cells BL21 were used for expression of the protein. IPTG was used as induction reagent for overexpression. Pure...
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Preparation of rat NK cell receptors using HEK293T expression system.
Celadová, Petra ; Bezouška, Karel (advisor) ; Půta, František (referee)
Natural killer cells play a significant role in the immune response against tumor and infected cells. NK cells express a wide variety of surface receptors, including NKRP1, a C-type lectin-like family of both activating and inhibitory receptors. Recently, ligands have been found for some of these previously orphan molecules, some of them lying within the same family. This is also the case of rat Clr-b as a cognitive ligand for rat NKRP1B. It has been shown that in rat, this inhibitory NKRP1B-Clr-b mutual receptor system is subverted by rat cytomegalovirus protein RCTL, a viral version of Clr-b, which serves as a decoy ligand for NK cells. The aim of my diploma thesis was cloning and production of the above mentioned C-type lectin-like proteins based on transient transfection of HEK293T cell line in a suspension culture. This expression system allows us not only to obtain proteins of our interest with a satisfactory yield but also in their native conformation, removing the need for time consuming and often fruitless refolding procedures required in case of using the E. coli expression system. Success was achieved in case of Clr-b and NKRP1B receptors from both WAG and SD strains. Proteins were purified using IMAC followed by gel filtration, identified by mass spectrometry and characterized by disulfide...
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Regulation of Cathepsin D Activity and Activation
Máša, Martin
PhD Thesis ABSTRACT REGULATION OF CATHEPSIN D ACTIVITY AND ACTIVATION Martin Máša supervisor: Michael Mareš Department of Biochemistry Faculty of Science Charles University Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic PRAGUE 2009 Introduction Cathepsin D (CD) is an aspartic peptidase located in the lysosomes of all mammalian cells, its main role is catabolic degradation of proteins. More over CD is known to participate in a range of physiological processes such as apoptosis and tissue homeostasis, as well as in the regulation of angiogenesis and the production of peptidic antigens. The role of CD in pathophysiology is associated with several diseases such as Alzheimer's disease and cancer. Alzheimer's disease is a neurodegenerative disorder, which is generally considered to be the most common form of dementia. Progression of this disease is accompanied with the deposition of amyloid plaques (AP) in the brain, which leads to neurodegeneration. The AP is a fragment released from amyloid precursor protein (APP) cleaved by secretases1 . High levels of CD were found in cerebrospinal fluid of the Alzheimer's patients2 . It was demonstrated that CD is able to cleave APP and produce the pathogenic AP. A genetic polymorphism in the CD gene was reported, which changes...
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