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Laboratory diagnostics of Lyme disease
DOSTÁLOVÁ, Simona
Lyme borreliosis is the most common infectious disease that is transmitted by ticks infected with Borrelia burgdorferi bacteria (anthropozoonosis). The diagnosis of this disease is based on the detection of specific IgG and IgM class immunoglobulins by specific laboratory tests. This bachelor thesis focuses on the evaluation of the clinical condition of patients by the attending physician (see case reports) in accordance with the results of laboratory tests. The correct diagnosis is based on the clinical symptoms, anamnesis and laboratory findings of each patient, which is used to guide adequate antibiotic treatment. In this work a comparison of two methods used for detection of antibodies against Borrelia burgdorferi, the chemiluminescence immunoassay (CLIA) method and the confirmatory Western blotting method used in the Clinical Laboratory of DIA-GON MP, s.r.o. in Cheb, is performed. The CLIA method provides rapid and sensitive screening for IgG and IgM antibodies, while Western blotting serves as a confirmatory test with higher specificity. The evaluation of the results and their interpretation are based on the detection of specific antibodies against Borrelia by Western blotting. The last part of the bachelor thesis is a search of an article from the Czech Hydrometeorological Institute, which cooperates with the State Institute of Health with the support of the Ministry of Health of the Czech Republic. It discusses tick activity in relation to their seasonal occurrence. On the basis of this article, I make an assessment of the seasonal occurrence of Lyme borreliosis in 2015-2017 with respect to the average temperatures in that period.
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Isolation and purification of nucleic acids using magnetised micro- and nanoparticles
Dostálová, Simona ; Adam, Vojtěch (referee) ; Kizek, René (advisor)
Nucleic acids are a completely unique component of every organism. They determine the appearance, behaviour or disposition to a certain disease. Despite their importance, there is still a lot of information unknown. In order to study the nucleic acids, they need to be purified from other components of the organism. The routine methods of isolation and purification are often laborious and time consuming; therefore experiments with magnetic separation are carried out. This thesis deals with magnetic micro- and nanoparticles and their application in this field. General conditions of this isolation are summarized in the thesis. Detection methods of nucleic acids are listed, focusing on electrochemical methods. A procedure for isolation and purification of nucleic acids using magnetic particles and square-wave voltammetry is described. This procedure was modified in experimental part of the thesis to achieve the maximum yield. Partial steps including washing, immobilization and elution were optimized. Results showed that the phosphate buffer was optimal for washing of beads before immobilization of nucleic acids on their surface. The optimal immobilization was carried out for 5 minutes at 20 °C and microtube needed to be firmly shaken during the process. Immobilization solution composed of 0.1M Na2HPO4 + 0.1M NaH2PO4, 0.6M guanidinium thiocyanate, 0.15M Trizma base adjusted by HCl on pH 7.5 and 2.5M CsCl. 5M NaCl was optimal for washing of the complex “beads-nucleic acid” after the immobilization. Elution was carried out for 15 minutes at 99 °C and the highest yield was obtained using elution solution composed of Tris-EDTA pH at 9.1
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Nanotransporters for theranostics
Dostálová, Simona ; Adam,, Vojtěch (referee) ; Kizek, René (advisor)
Master thesis deals with the use of bacteriophage as a theranostic drug nanocarrier. The term theranostics is used in recent years for systems that allow connecting of diagnostics, targeted drug delivery and monitoring of patient’s response to administered treatment in a single modality. These systems are very suitable especially with heterogeneous diseases, such as cancer. Nowadays, the treatment of cancer has often severe side effects to the patient’s body, which lowers his capability to fight the disease. Theoretical part of this work is focused on the properties of viral capsids, proteins and inorganic materials as drug nanocarriers. In practical part of this work, different methods for cultivation of bacteriophage are compared, both in liquid and solid medium and with different concentrations of the maltose, trough whose receptors bacteriophage is able to enter the host cell. Optimal was cultivation with 0.2% maltose in solid medium. Practical part is focused mainly on the use of bacteriophage as a nanocarrier for cytotoxic drug doxorubicin. Bacteriophage was able to encapsulate all applied concentrations of doxorubicin (0; 12.5; 25; 50; 100 and 200 g/ml), which was proved using fluorescent detection. Different times of encapsulation (2; 4; 8 and 12 hours) were studied. Optimal time was 2 hours. Encapsulation properties of bacteriophage were compared to apoferritin. Bacteriophage was able to encapsulate 4× higher concentrations of doxorubicin and its release during rinsing with water was 10× lower compared to apoferritin. This work concludes that bacteriophage is a very suitable platform for targeted drug delivery in theranostics.
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Craniofacial Abnormalities in Sleep Apnoea Syndrome
Dostálová, Simona ; Šonka, Karel (advisor) ; Čáp, Jan (referee) ; Peterka, Miroslav (referee)
The thesis is based on a series of cephalometric studies of patients with SAS, acromegalic patients with or without SAS as well as control groups. The studies presented in this thesis produce following results: There are important differences in orofacial skeleton and soft tissue of nasopharynx measured by cephalometry in the patients with sleep apnoea compared to the control group of women and men. Proved skeleton changes (increased lower gonion angle, increased anterior facial heights, decreased depth of the upper face) and elongated soft palate predisposes patients to narrowing of upper airways in oropharyngx, which significantly contributes to development of SAS. Cephalometric differences in the size of the soft palate, the rotation of the mandible and the size of lower gonion angle can be found in men with mild and severe sleep apnoea. We conclude that the severity of the SAS depends on the degree of changes in the orofacial skeleton and adjacent soft tissues. The results, which reveal a significant differences in BMI and in the circumference of the neck between groups with mild and severe SAS, confirm the idea that the most important predisposing factor for SAS is obesity, in particular deposition of adipose tissue in the posteriolateral parapharyngeal space. Patients with acromegaly (of both sexes)...
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Craniofacial Abnormalities in Sleep Apnoea Syndrome
Dostálová, Simona ; Šonka, Karel (advisor) ; Čáp, Jan (referee) ; Peterka, Miroslav (referee)
The thesis is based on a series of cephalometric studies of patients with SAS, acromegalic patients with or without SAS as well as control groups. The studies presented in this thesis produce following results: There are important differences in orofacial skeleton and soft tissue of nasopharynx measured by cephalometry in the patients with sleep apnoea compared to the control group of women and men. Proved skeleton changes (increased lower gonion angle, increased anterior facial heights, decreased depth of the upper face) and elongated soft palate predisposes patients to narrowing of upper airways in oropharyngx, which significantly contributes to development of SAS. Cephalometric differences in the size of the soft palate, the rotation of the mandible and the size of lower gonion angle can be found in men with mild and severe sleep apnoea. We conclude that the severity of the SAS depends on the degree of changes in the orofacial skeleton and adjacent soft tissues. The results, which reveal a significant differences in BMI and in the circumference of the neck between groups with mild and severe SAS, confirm the idea that the most important predisposing factor for SAS is obesity, in particular deposition of adipose tissue in the posteriolateral parapharyngeal space. Patients with acromegaly (of both sexes)...
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Isolation and purification of nucleic acids using magnetised micro- and nanoparticles
Dostálová, Simona ; Adam, Vojtěch (referee) ; Kizek, René (advisor)
Nucleic acids are a completely unique component of every organism. They determine the appearance, behaviour or disposition to a certain disease. Despite their importance, there is still a lot of information unknown. In order to study the nucleic acids, they need to be purified from other components of the organism. The routine methods of isolation and purification are often laborious and time consuming; therefore experiments with magnetic separation are carried out. This thesis deals with magnetic micro- and nanoparticles and their application in this field. General conditions of this isolation are summarized in the thesis. Detection methods of nucleic acids are listed, focusing on electrochemical methods. A procedure for isolation and purification of nucleic acids using magnetic particles and square-wave voltammetry is described. This procedure was modified in experimental part of the thesis to achieve the maximum yield. Partial steps including washing, immobilization and elution were optimized. Results showed that the phosphate buffer was optimal for washing of beads before immobilization of nucleic acids on their surface. The optimal immobilization was carried out for 5 minutes at 20 °C and microtube needed to be firmly shaken during the process. Immobilization solution composed of 0.1M Na2HPO4 + 0.1M NaH2PO4, 0.6M guanidinium thiocyanate, 0.15M Trizma base adjusted by HCl on pH 7.5 and 2.5M CsCl. 5M NaCl was optimal for washing of the complex “beads-nucleic acid” after the immobilization. Elution was carried out for 15 minutes at 99 °C and the highest yield was obtained using elution solution composed of Tris-EDTA pH at 9.1
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Nanotransporters for theranostics
Dostálová, Simona ; Adam,, Vojtěch (referee) ; Kizek, René (advisor)
Master thesis deals with the use of bacteriophage as a theranostic drug nanocarrier. The term theranostics is used in recent years for systems that allow connecting of diagnostics, targeted drug delivery and monitoring of patient’s response to administered treatment in a single modality. These systems are very suitable especially with heterogeneous diseases, such as cancer. Nowadays, the treatment of cancer has often severe side effects to the patient’s body, which lowers his capability to fight the disease. Theoretical part of this work is focused on the properties of viral capsids, proteins and inorganic materials as drug nanocarriers. In practical part of this work, different methods for cultivation of bacteriophage are compared, both in liquid and solid medium and with different concentrations of the maltose, trough whose receptors bacteriophage is able to enter the host cell. Optimal was cultivation with 0.2% maltose in solid medium. Practical part is focused mainly on the use of bacteriophage as a nanocarrier for cytotoxic drug doxorubicin. Bacteriophage was able to encapsulate all applied concentrations of doxorubicin (0; 12.5; 25; 50; 100 and 200 g/ml), which was proved using fluorescent detection. Different times of encapsulation (2; 4; 8 and 12 hours) were studied. Optimal time was 2 hours. Encapsulation properties of bacteriophage were compared to apoferritin. Bacteriophage was able to encapsulate 4× higher concentrations of doxorubicin and its release during rinsing with water was 10× lower compared to apoferritin. This work concludes that bacteriophage is a very suitable platform for targeted drug delivery in theranostics.
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