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On-chip and capillary electrophoresis for cancer research
Hájková, Tereza ; Babula, Petr (referee) ; Provazník, Ivo (advisor)
The thesis deals with cancer disease and its genesis. The prostate cancer is the second most dangerous disease in male population. It is very important to detect it in early stage. This thesis deals with the proteins which are or could be used as potential cancer markers. For example in current commonly used the prostate-specific antigen and newly the metallothionein. Metallothionein is a protein occuring in fauna and flora in four basic isoforms. There is a hypothesis that the isoform content or/and their ratio may have a diagnostic implication and could be used in clinical practise. For this reason they are studied and developed methods for specific and sensitive analysis of individual isoforms. In this work, capillary electrophoresis is used for the separation of the isoforms. The influence of the parameters such as type of background electrolyte, its pH and separation voltage etc. on the final separation are discussed.
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Differential Gene expression using a negative binomial model
Janáková, Tereza ; Tkacz, Ewaryst (referee) ; Abo Khayal, Layal (advisor)
Hlavním cílem této diplomové práce je analýza diferenciální exprese genů na základě negativního binomického modelu. Úvodní část je věnována teoretickému základu, pojednává o sekvenování RNA, sekvenování nové generace, výhodách a možném využití, formátu fastQ aj. Následující část už se zabývá samotnou praktickou částí, zde byl vybrán vhodný set genů, které budou později analyzovány a příslušná data byla stažena. Tato data byla zarovnána k lidskému genomu verze 37 Burrowsovou-Wheelerovou transformací s využitím bowtie mapovače, byly tak vytvořeny soubory ve formátu SAM. Toto soubory dat byly později setříděny pomocí nástroje SAMtools. Následně byly v programovém prostředí Matlab (verze R2013b) vytvořeny anotované objekty genů s využitím služby Ensembl´s BioMart. Dále byla určena genová exprese a byly odhadnuty faktory velikosti knihovny. Na závěr byly odhadnuty parametry negativního binomického rozložení a byla vyhodnocena diferenciální exprese genů.
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Cancer Immunotherapy exploiting engineered antibody fragments against prostate-specific membrane antigen
Das, Gargi ; Bařinka, Cyril (advisor) ; Vaněk, Ondřej (referee) ; Ormsby, Tereza (referee)
Prostate cancer (PCa) remains a leading cause of male cancer-related mortality, necessitating thus the development of novel therapeutic approaches as conventional treatments have limited efficacy. Prostate-specific membrane antigen (PSMA) is an established biomarker for both imaging and therapy of PCa, as it is highly upregulated in neoplastic PCa tissues and metastatic castration- resistant prostate cancer. Consequently, immunological targeting of PSMA has gained significant attention as a therapeutic platform for the management of the disease. The thesis is focused on engineering of antibody fragments and fusion proteins derived from the high affinity anti-PSMA 5D3 monoclonal antibody that can be used as immune cell engagers to target and eliminate PSMA-positive cells. To this end, we engineered 5D3 single chain variable fragments (scFv) that were subsequently fused to anti-CD3 scFv and CP33 sequences, creating thus immune cell engagers targeting T-cells (BiTE) and monocytes (5D3-CP33), respectively. The engagers were expressed in insect cells, purified to homogeneity and their biophysical and functional characteristics evaluated using size exclusion chromatography, differential scanning fluorimetry, ELISA and flow cytometry. Ensuing cell-based assays revealed that both BiTE and 5D3-CP33 can...
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The diagnostic and prognostic ability of selected serum and urinary markers of prostate cancer
Do Carmo Silva, Joana Isabel ; Veselý, Štěpán (advisor) ; Čapoun, Otakar (referee) ; Fedorko, Michal (referee)
The diagnostic and prognostic ability of selected serum and urinary markers of prostate cancer Abstract Serum prostate specific antigen (PSA) is the only widely approved marker in prostate cancer (PC) diagnosis and follow up after treatment. Its role has remained controversial due to lack of specificity and the risk of overdiagnosis of insignificant PC. The aim of this work was to explore promising markers of PC and to improve current patient stratification to adjuvant treatment. Three main studies were performed using different media (urine and serum). The first study included the evaluation of Engrailed-2 (EN2) - a urinary marker of interest - in 90 patients with localized PC, 30 healthy controls, and 40 patients indicated for prostate biopsy. The second study evaluated 205 men with high-risk PC-features who underwent radical prostatectomy (RP) and were subject to a strict follow-up protocol of ultrasensitive PSA (UPSA) at close time intervals. The ability of particular measurements to predict biochemical recurrence (BCR) and thus the need for adjuvant therapy was assessed using the area under the curve (AUC) and a stratification model was created. The third study involved 128 patients who underwent RP. PSA and its serum isoforms normally used in the diagnostic context were evaluated both preoperatively...
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Theragnostics - new methods in nuclear medicine
PROCHÁZKOVÁ, Michaela
The main topic of the bachelor thesis is theragnostics - new methods in nuclear medicine. Theragnostics is a personalized treatment strategy that combines diagnostics with therapy and finds application primarily in the treatment of cancer. Theragnostics as a medical field is still evolving. The aim of this work was to map the practical use of current indications of theragnostic methods in nuclear medicine. The theoretical part of the thesis defines the concept of theragnostics and theragnostic methods containing nanoparticles and radionuclides. There is described the use of radionuclide theragnotics in clinical practice, especially in the treatment of thyroid gland, neuroendocrine tumors and prostate cancer. In this part of the thesis there are also clinical studies for theragnotic practice. Furthermore in the theoretical part of the thesis is maped the practical use of current indications of theragnotic methods, as well as possible future perspectives of theragnotic treatment. The theoretical part is processed from professional publications and articles. The practical part of the work focuses on the awareness of radiology assistants about this new treatment method. The respondents were radiology assistants across hospitals. There were 32 respondents involved to complete the research questions for the questionnaire survey. The evaluation from the collected data of the questionnaire survey is in the form of the percentage success of radiological assistants. The overall concept of this bachelor's thesis can serve as a study material for university students in the field of radiology assistant.
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Magnetic resonance imaging of prostate and the success rates in statistics of guided biopsy of the prostate
HEFLER, Jakub
A carcinoma of a prostate is a very serious and common disease in the Czech republic and even in the whole world. It's incidence in 2018 was 62,5 in 100 000 across the Czech republic. To this day incidence and mortality slowly decreases. Diagnosis of a prostate carcinoma is started with a palpating examination per rectum. Then we determine a patient's PSA. If the PSA level is high, the patient is indicated for a biopsy od the prostate which is done with an ultrasound navigation. For more precise biopsy we use an image fusion. This means that the urologist, which is doing the biopsy, fuses an MRI image with the ultrasound. During the biopsy 10-15 samples are taken and they're histologically examined. A pathologist then determines a Gleason score of the carcinoma which takes values of 1-5 and it shows a differentiation and type of the carcinoma. Medical professionals also measure the risk of having a prostate carcinoma with PI-RADS 2.1 system. This system also has a scale of 1 to 5. A guided biopsy is usually done to patients with PI-RADS 4 or 5. For this bachelor thesis I used data from an urology department of Nemocnice České Budějovice. I took data about patients that were examined from April of 2019 to June of 2021. During that time 288 patients were examined. 45 patients that went through a MR guided biopsy were randomly chosen and their data were statistically processed. Goals of this work were: - Creating an informational text that contains anatomy and fysiology of the prostate, a mechanism of the MRI and a description on a guided biopsy of a prostate. - Determination of a statistical succes rate of an MRI guided biopsy of a prostate. Also there were hypothesis: H1: If it happens that laboratory and hystological results are not corresponding, majority of the patients are indicated for an MRI guided biopsy of the prostate. H2: At least 80% of patients have a positive result for a prostate carcinoma after an MRI navigated biopsy of a prostate. An informational text was created so the fist goal was fulfilled. Goal number 2 was fulfilled also because a statistical rate of positive and negative results of the guided biopsy were determined. This rate is 73% positive and 27% negative findings so it is necessary to reject a hypothesis H2. We can accept a hypothesis H1 according to information from MUDr. Černý that takes care of the MRI guided biopsies in Nemocnice České Budějovice.
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Splice variants of the gene coding for GCPII and their role in cancer development
Jindrová, Helena ; Konvalinka, Jan (advisor) ; Liberda, Jiří (referee)
Alternative splicing is a mechanism of generating distinct proteins that are encoded by the same gene. These proteins differ in amino acid sequence, overall structure and function. Splicing dysregulations have been shown to be implicated in several pathologic processes including cancer. For example, non-physiological splicing of osteopontin was proved to play a key role in cell progression of breast cancer. Glutamate carboxypeptidase II (also called prostate specific membrane antigen, PSMA) is present in both normal prostate and prostate cancer. Several splice variants of PSMA have been described and it has been suggested that the overexpression of some of them could be involved in the progression of prostate cancer. Nevertheless, more detailed investigation of each of the PSMA splice variant in terms of their occurrence in prostate cancer cells remains to be performed. This thesis focuses on the exploration of the expression of PSMA splice variants with deleted exons 6 and 18 in samples of a cell line derived from human prostate cancer, benign prostate hyperplasia and prostate cancer. For this purpose, RT-PCR was utilized to determine the ratio of deletions of exons 6 and 18 in cDNA of the prostate specific membrane antigen. Furthermore, the ratio of deletions of exon 6 and 18 was determined in...
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Antibody derivatives for the detection of human glutamatecarboxypeptidase II
Bělousová, Nikola ; Bařinka, Cyril (advisor) ; Pavlíček, Jiří (referee)
Prostate cancer is one of the most common human malignancies and, consequently it is critical to develop appropriate diagnostic and therapeutic tools. Glutamate carboxypeptidase II (GCPII) is currently being considered one of the most important prostate cancer markers due to its tissue- specific expression. Whereas in healthy prostatic tissue the expression levels of GCPII are low, the transformation into the tumor is associated with the substantial increase of GCPII expression, with the highest levels observed in androgen-independent metastatic tumors. GCPII is thus considered a promising marker for early phase as well as advanced metastatic stages of prostate cancer. Current research is focused on the development of highly sensitive and specific reagents that allow detection of small amounts of GCPII, for example in early stages of cancer. Antibody derivatives are promising molecules for this purpose because they have high affinity and specificity and minimum negative side effects. Protein engineering is a prefered approach for preparation of various antibody molecules that differ in size, binding properties, stability, solubility, and production means. Different types of derivatives are being developed for medical needs such as in vitro diagnosis, therapy, and in vivo imagingSmall molecular...
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Characterisation of the cell line TRAMP-C2 side population, mouse model of prostate cancer
Žlabová, Anna ; Reiniš, Milan (advisor) ; Šmahel, Michal (referee)
Side population is a minor subpopulation (SP) of some cell lines, exporting staining dye Hoechst 33342 out of their cytoplasm. It is discussed as a possible source of "cancer stem cells", "tumour initiating cells" or "metastasis initiating cells". However, broad literature suggest, that stemness and other privileged properties of SP are very variable between different cell types, cell lines and stage of disease. Cell lines TRAMP are the only widely available murine models for testing of prostate cancer therapy. We noticed in literature a mention about existence of 1-2% of cells constituting side population, but detailed characteristic have not been described until now. In this diploma thesis, we worked on characterisation of SP of the TRAMP-C2 cell line in comparison to other cells (nonSP). In the first part, we compared stem properties of SP and nonSP. We started with checking the existence of SP by its verapamil sensitivity. Using mRNA analysis, we showed that neither SP nor nonSP have increased c-Kit expression and that there are no differences in Bmi-1 expression. We found that SP is heterogenic mixture of CD24-CD44-, CD24-CD44+ and CD24+CD44+ cells, while nonSP is almost solely CD24-CD44+. We documented that SP and nonSP returned back to original SP ratio during cultivation. Then we showed on...
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