Characterising transcription factor binding sites (TFBS) is an important problem in bioinformatics, since predicting binding sites has many applications such as predicting gene regulation. ChIP-seq is a powerful in vivo method for generating genome-wide putative binding regions for transcription factors (TFs). CentriMo is an algorithm that measures central enrichment of a motif and has previously been used as motif enrichment analysis (MEA) tool. CentriMo uses the fact that ChIP-seq peak calling methods are likely to be biased towards the centre of the putative binding region, at least in cases where there is direct binding. CentriMo calculates a binomial p-value representing central enrichment, based on the central bias of the binding site with the highest likelihood ratio. In cases where binding is indirect or involves cofactors, a more complex distribution of preferred binding sites may occur but, in many cases, a low CentriMo p-value and low width of maximum enrichment (about 100bp) are strong evidence that the motif in question is the true binding motif. Several other MEA tools have been developed, but they do not consider motif central enrichment. The study investigates the claim made by Zhao and Stormo (2011) that they have identified a simpler method than that used to derive the UniPROBE motif database for creating motifs from protein binding microarray (PBM) data, which they call BEEML-PBM (Binding Energy Estimation by Maximum Likelihood-PBM). To accomplish this, CentriMo is employed on 13 motifs from both motif databases. The results indicate that there is no conclusive difference in the quality of motifs from the original PBM and BEEML-PBM approaches. CentriMo provides an understanding of the mechanisms by which TFs bind to DNA. Out of 13 TFs for which ChIP-seq data is used, BEEML-PBM reports five better motifs and twice it has not had any central enrichment when the best PBM motif does. PBM approach finds seven motifs with better central enrichment. On the other hand, across all variations, the number of examples where PBM is better is not high enough to conclude that it is overall the better approach. Some TFs bind directly to DNA, some indirect or in combination with other TFs. Some of the predicted mechanisms are supported by literature evidence. This study further revealed that the binding specificity of a TF is different in different cell types and development stages. A TF is up-regulated in a cell line where it performs its biological function. The discovery of cell line differences, which has not been done before in any CentriMo study, is interesting and provides reasons to study this further.

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

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Acute myeloid leukemia(AML) is the most common acute leukemia among adults in the United states with approximately 19,940 people being diagnosed of this disease in 2020 and 11,180 deaths. It is a heterogenous group of malignancy characterized by clonal expansion of blast with myeloid lineage in the bone marrow, peripheral blood and/or other tissues. Our patient is a 79-year-old male who presented to the hospital with reports of sharp, throbbing low back pain for one month, moderately controlled with pain medications. He reported 5 lb. weight loss with decreased appetite over one month but denied other constitutional symptoms. MRI Lumbar spine revealed multiple foci of marrow signal abnormality compatible with extensive metastatic disease. CT chest, abdomen and pelvis did not show any lesions concerning for primary or metastatic malignancy. CBC revealed normal WBC count, platelet count and hemoglobin level (with macrocytosis, MCV 104.7). Initial work up including Vitamin B12 and folic acid level, TSH, SPEP/IFE, serum light chain ratio and quantitative immunoglobulins were within normal limits. Pathology from a CT guided bone biopsy of the L spine lesion was concerning for high grade myeloid neoplasm. Patient had a bone marrow biopsy done at another hospital which was read as most consistent with acute myeloid leukemia (AML) with monocytic differentiation, with findings of hypocellular marrow, extensive fibrosis with focal areas of large clusters of immature cells, positive for MPO, CD33, CD43 and CD 56, Ki-67 of 60-80%. Cytogenetics showed an abnormal male karyotype with trisomy 8. FISH was negative for other AML or MDS related abnormalities. Given the above findings of AML and advanced age, patient was started on treatment with hypomethylating agent Decitabine along with BCL-2 inhibitor, Venetoclax. A repeat bone marrow biopsy after two cycles of the above regimen revealed progressive disease with extensive fibrosis and 80-90% blast on a core biopsy sample. Due to poor response to above regimen, lack of effective treatment options in older patients with AML and declining functional status, decision was made to pursue best supportive care. AML usually presents with symptoms of fevers, fatigue, dyspnea or bleeding. Skeletal lesions are usually associated with a diagnosis of multiple myeloma, or other solid organ malignancies and rare in AML. Extra medullary involvement of AML is known to happen in 2.5%-9% of patients and is termed as Myeloid Sarcoma. Due to the low incidence, prospective study data is limited. This entity is treated similarly to AML, depending on risk stratification by cytogenetics, age and targetable mutations which also govern its prognosis. This case highlights the importance of increased awareness and high index of suspicion among medical providers regarding this atypical presentation of AML since if missed or misdiagnosed could delay treatment and lead to poor outcomes.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Miškovčíková Supervisor: RNDr. Jana Maixnerová, PhD. Title of diploma thesis: Determination of organ toxicity of BRAF inhibitors in vitro. Malignant melanoma is one of the most serious skin diseases today. Therapy of advanced melanoma is difficult and often ineffective. BRAF inhibitors (dabrafenib and vemurafenib) have dramatically changed the results of melanoma treatment in the last few years. BRAF inhibitors are one of the most effective drugs against melanoma, but their clinical application is largely limited by drug resistance. Available clinical studies have shown an adverse nephrotoxic effect of BRAF inhibitors, but information on its mechanism is limited. Published studies further suggest that the toxic effect of BRAF inhibitors is primarily directed to podocytes located in the glomerular membrane. Thus, the aim of our study was to assess the cytotoxic effect of BRAF inhibitors on selected model renal cells in vitro in order to confirm the renal target toxicity. The main objective of the study was to analyse whether the nephrotoxic effect of BRAF inhibitors is specifically limited to podocytes or whether it can damage other renal cells. The experiments were performed on human cell lines...

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Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Bc. Marie Zádrapová Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Determination of renal toxicity of antineoplastics in vitro BRAF inhibitors are important antineoplastics. They work on the principle of inhibition of certain types of protein kinases and turned out to be very efficient for the treatment of melanoma. One of their disadvantages is relatively early onset of resistance; thus, it is important to look for new combinations of drugs that are already in use or work on the development of new structures with similar inhibition efficacy on melanoma cells. Encorafenib and its combination with binimetinib have been shown to be very promising drugs from the group of BRAF inhibitors, however, potential renal toxicity may be a therapeutic limitation. This thesis was focused on the determination of in vitro cytotoxicity of encorafenib on different types of renal cells in three time intervals and on its comparison with two drug standards - amphotericin B and paracetamol. Three types of morphologically and functionally different kidney cells (PODO / TERT256, HK-2 and HEK293) were used for this purpose. The cytotoxic potential was measured by colorimetric method CellTiter 96®...

Multiple Myeloma is cancer of plasma cells and is known to be highly invasive. Multiple Myeloma makes up 1% of cancer diagnosis in western countries and affects men more predominantly than women. The American Cancer Association estimates that 32,110 new cases will be diagnosed in the United States in 2019. Lenalidomide is one of the main therapies used for multiple myeloma patients, but it has toxic side effects such as thrombocytopenia, neutropenia, and anemia. The purpose of the study is to investigate new cytotoxic agents for the treatment of multiple myeloma. In addition to lenalidomide alone, this study examined the effects of doxycycline alone and in combination with lenalidomide. Lenalidomide cell cultures were treated at concentrations from 0.5μM to 10μM on untreated 24 well plates and doxycycline concentration ranging from 10μM-80μM. Following incubation, cell viability was tested using MTT assay and the samples were analyzed using spectrophotometry. When compared to lenalidomide, doxycycline monotherapy showed a greater decrease in overall cell viability in preliminary results. Our results show that there is benefit of using 10μM of Doxycycline at higher concentration of 5μM and 10μM of lenalidomide. The potential decrease in the concentration of lenalidomide used by adding doxycycline, may reduce the toxic side effects of lenalidomide. Further studies are necessary to confirm these preliminary results and investigate the mechanism of action in order to determine optimal combinations of these drugs.

Breast cancer is the most commonly occurring cancer worldwide, more than 2.26 million new cases occurred in women in 2020. Treatment for breast cancer is normally individualized to the patient based on the presence of different receptors, these receptors include HER2, progesterone, and estrogen. The presence of these receptors generally comes with an amenable prognosis, and a wide array of available treatments. There are types of breast cancers that do not have any of these receptors and tend to be much more aggressive. This type of cancer is called triple negative and represents about 10 percent of all breast cancer occurrences in the United States. The lack of receptors makes this type of cancer extremely difficult to treat and generally comes with a poor prognosis. In the present study, we used triple negative breast cancer cell line MDA-MB231, which is known to use actin remodeling to evade immunologic response. These cells were treated with two novel, structurally similar flavonoids, CT1 and CT3 derived from an ethnobotanically recognized species of Chromolaena. CT1 and CT3 are extracted from the leaves of Chromolaena tacotana and then isolated and purified by chromatography. These compounds are used to treat cancer cells, at different concentrations that include 5, 10, 20, 40 and 80mM. MTT assays are used to determine their effect on cell viability, and the mechanism of action was analyzed by immunoblotting and TUNEL.. CT1 has a significantly stronger inhibitory effect on MDA-MB231 cell viability as compared to CT3. Preliminary analysis of the mechanism of action of CT1 has revealed that it neither follows the canonical intrinsic apoptotic pathway nor the extrinsic pathway that involves the activated form of c-JUN. By up-regulating actin, triple negative breast cancer is able to evade immunologic response and cancer treatment. CT1, a novel flavonoid extracted from the leaves of Chromoleana tacotana has shown cytotoxic effects against triple negative breast cancer cells effectively bypassing the actin response. The mechanism of action is currently under study.

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