Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Jesika Štorkánová Supervisor: RNDr. Eva Novotná, Ph.D. Title of diploma thesis: Study of the cytotoxicity of selected chemotherapeutics for the treatment of leukemia in human tumor cell lines Leukemia represents a diverse group of malignant diseases with a hematopoietic disorder with different prognoses. As the incidence of patients with leukemia is increasing, is an effort to establish the treatment that will lead to successful therapy. One of the basic approaches to the treatment of leukemias is chemotherapy. Today it is known that the effectiveness of chemotherapy is influenced by a number of factors which can significantly affect the treatment strategy and thus decide on the outcome of the treatment itself. An important approach in chemotherapy is the selection of cytostatics with maximum efficacy for oncological disease and elimination cytostatics to which the cells are resistant based on the findings in in vitro conditions. The aim of this diploma thesis was to determine the inhibitory effects of in vitro selected chemotherapeutics in cell tumor lines. For determine the inhibitory effect, HCT116, HepG2 and HL-60 cell lines were selected using a colorimetric method based on the...

Student Number : 9906751K - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / In addition to its crucial role in cell adhesion, β-catenin is also known to augment gene expression by forming a complex with lymphoid enhancer factor/T-cell factor in the nucleus. Unregulated β-catenin expression and/or its increased nuclear presence can lead to abnormal cell proliferation, tumour invasion and metastasis. Pertinent is the fact that the actin cytoskeleton is central to the translocation of several nuclear proteins. This study investigated whether the actin cytoskeleton influences the nuclear translocation of β-catenin in human oesophageal squamous cell carcinoma (HOSCC), a metastatic disease of common occurrence in South Africa. Disruption of the actin cytoskeleton of five moderately differentiated HOSCC cell lines, with cytochalasin D (cytoD), showed that the nuclear β-catenin level was unaltered in SNO, WHCO1 and WHCO5, but decreased in WHCO3 and WHCO6. CytoD treatment did not affect the cytoplasmic/membrane β-catenin level in these cell lines. Further examination of the possible association between the actin cytoskeleton and nuclear β-catenin translocation, required the design and stable transfection, of a vector containing full-length human β-catenin cDNA into one of the HOSCC lines. Stimulation of exogenous β-catenin expression in transfected WHCO1 cells did not increase cellular β-catenin level, nor did the stimulation of endogenous β-catenin expression with DMSO. In most cases (SNO, WHCO1 and WHCO5) the nuclear distribution of β-catenin in HOSCC is independent of a functional actin cytoskeleton, nonetheless there are some exceptions (WHCO3 and WHCO6). The observed variation within the HOSCC lines is possibly due to specific underlying event/s particular to the cell line. The stable level of β-catenin expression could be a consequence of regulatory pathways in WHCO1 compensating for the induced imbalance of β-catenin expression.

Les voies de signalisation canoniques traditionnelles aident à comprendre l'ensemble des processus de signalisation à l'intérieur de la cellule. Les données phosphoprotéomiques à grande échelle donnent un aperçu des altérations entre différentes protéines dans différents contextes expérimentaux. Notre objectif est de combiner les réseaux de signalisation traditionnels avec des données de séries temporelles phosphoprotéomiques complexes afin de démêler les réseaux de signalisation spécifiques aux cellules. Côté application, nous appliquons et améliorons une méthode de séries temporelles caspo conçue pour intégrer des données phosphoprotéomiques de séries temporelles dans des réseaux de signalisation de protéines. Nous utilisons une étude de cas réel à grande échelle tirée du défi HPN-DREAM BreastCancer. Nous déduisons une famille de modèles booléens à partir de données de séries temporelles de perturbations multiples de quatre lignées cellulaires de cancer du sein, compte tenu d'un réseau de signalisation protéique antérieur. Les résultats obtenus sont comparables aux équipes les plus performantes du challenge HPN-DREAM. Nous avons découvert que les modèles similaires sont regroupés dans l'espace de solutions. Du côté informatique, nous avons amélioré la méthode pour découvrir diverses solutions et améliorer le temps de calcul. / Traditional canonical signaling pathways help to understand overall signaling processes inside the cell. Large scale phosphoproteomic data provide insight into alterations among different proteins under different experimental settings. Our goal is to combine the traditional signaling networks with complex phosphoproteomic time-series data in order to unravel cell specific signaling networks. On the application side, we apply and improve a caspo time series method conceived to integrate time series phosphoproteomic data into protein signaling networks. We use a large-scale real case study from the HPN-DREAM BreastCancer challenge. We infer a family of Boolean models from multiple perturbation time series data of four breast cancer cell lines given a prior protein signaling network. The obtained results are comparable to the top performing teams of the HPN-DREAM challenge. We also discovered that the similar models are clustered to getherin the solutions space. On the computational side, we improved the method to discover diverse solutions and improve the computational time.

"Sin Nombre virus is a hantavirus first recognized in New Mexico in 1993. This virus is responsible for causing Hantavirus Pulmonary Syndrome, an acute, life threatening illness characterized by pulmonary edema, capillary leaking, and extreme respiratory distress. CD8+ cytotoxic T-cell lines specific for Sin Nombre virus were isolated from the peripheral blood mononuclear cells (PBMC) of a donor (NM3) who was naturally infected with the Sin Nombre virus, and has survived hantavirus pulmonary syndrome (HPS). Cytotoxic T lymphocyte (CTL) assays showed that one of these cell lines, 10K, specifically recognizes a nine amino acid epitope, TAHGVGIIP (amino acids 664-672 of the precursor GPC protein), which is located in the G2 protein after cleavage. Another cell line, 10c27, specifically recognized an eight amino acid epitope, AHGVGIIP (amino acids 665-672 of the precursor GPC protein), located in the G2 protein after cleavage. Using polymerase chain reaction (PCR) and CTL assays, the recognition of these epitopes was shown to be restricted by the B35.01 Class 1 human leukocyte associated antigen (HLA) allele. This information will be useful in creating a vaccine for use in immunizing people against the Sin Nombre hantavirus, as well as elucidating the pathogenesis of this disease. "

While many investigations have been performed to establish a better understanding of the effects that magnetic fields and nanoparticles have on cells, the fundamental mechanisms behind the interactions are still yet unknown, and investigations on concurrent exposure are quite limited in scope. This study was therefore established to investigate the biological impact of concurrent exposure to magnetic nanoparticles and extremely-low frequency magnetic fields using an in-vitro CHO-K1 cell line model, in an easily reproducible manner to establish grounds for further in-depth mechanistic, proteomic, and genomic studies. Cells were cultured and exposed to 10nm Fe3O4 nanoparticles, and DC or low frequency (0Hz, 50Hz, and 100Hz) 2.0mT magnetic fields produced by a Helmholtz coil pair. The cells were then observed under confocal fluorescence microscopy, and subject to MTT biological assay to determine the synergistic effects of these concurrent exposures. No effects were observed on cell morphology or microtubule network; however, cell viability was observed to decrease more drastically under the combined effects of magnetic field and nanoparticle exposures, as compared to independent exposures alone. It was concluded that no significant difference was observed between the types of magnetic fields, and their effects on the nanoparticle exposed cells, but quite clearly there are deleterious synergistic effects of these concurrent magnetic field and nanoparticle exposure conditions.

Finally, the pharmacodynamic effects of propranolol powder formulation with different buffering were carried out in two healthy male subjects. The maximal reduction in heart rate was found at the saliva pH of 7.6, which corresponded to the pHmax of propranolol. A buffered propranolol sublingual tablet was then prepared to achieve the saliva pH around 7.6. The preliminary investigation confirmed that the sublingually administrated buffered propranolol tablet produced a faster and more pronounced heart rate reduction than the non-buffered commercial propranolol tablet. / Firstly, the use of the HO-1-u-1 cell culture for screening sublingual drug delivery was validated. The cells were seeded on cell culture inserts. The integrity of cell layers, inter-passage variation and directionality were assessed by measuring the resistance and the permeability of standard markers, beta-blockers and calcium channel blockers. The effect of pH, osmolarity and a permeation enhancer (GDC) were also studied. The results showed that HO-1-u-1 cells grown on inserts formed stratified and epithelial-like structure that preserved the typical histological feathers of the normal human sublingual epithelium. The maximal integrity was reached in 23 days. The Papp of beta-blockers and calcium channel blockers ranged from 2.89+/-0.17 x 10 -6 cm/s to 6.37+/-0.37 x 10-6 cm/s. The permeability of selected beta-blockers under different pH, osmolarity and GDC revealed that enhancing effects were significant for hydrophilic compounds but less for lipophilic compounds. / Secondly, fresh porcine sublingual mucosa was prepared and compared to the cell line model. Good correlations were obtained for both the Papp of beta-blockers and the enhancement ratios of pH and GDC between the two models. / The aims of the present study are (1) to develop and validate a human sublingual epithelial cell line model and (2) to demonstrate the application in sublingual development of cardiovascular drugs. / Thirdly, the steady-state flux (Jss) at various pH levels were measured. Results show that saturated propranolol solution at pH 7.0--7.6 resulted in a much higher Jss than the solution at other pHs. These data led to the development of theoretical equations for predicting the optimum pH (pHmax) for ionizable compounds. The calculation fitted well with the experimental data. / Wang Yanfeng. / Advisers: Moses S. S. Chow; Zhong Joan Zuo. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 184-). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Rett syndrome (RTT) is a severe neurological disorder that affects approximately 1:10000 girls. Classical RTT is defined by a developmental regression phase and subsequent stabilisation of diagnostic criteria, which include partial or complete loss of spoken language, dyspraxic gait and stereotypic hand movements such as hand mouthing. RTT is a monogenic disorder, with the majority of cases being due to loss-of-function mutations in MeCP2 (methyl-CpG binding protein 2). Due to this clear genotype-phenotype link multiple RTT mouse models have been used to elucidate the molecular details, and consequent neuropathogenesis, of this complex neurological disease, as well as for the development of potential therapeutics for RTT. However, as the molecular details become clearer, the need for a simpler model system becomes evident. Human induced pluripotent stem cells (hiPSCs) generated from RTT patient fibroblasts are an option; however the handling of these cells is laborious, time-consuming and expensive and they often differentiate into a heterogeneous population of cells. To explore an alternative human model system I have been genetically engineering and experimenting with the human dopaminergic LUHMES cell line. LUHMES cells are an immortalised pre-neuronal cell line derived from an 8-week old, female foetus and can readily be differentiated into a homogeneous population of mature, electrically active neurons in just one week. In this thesis I have assessed the phenotypic properties of the wild-type cell line, demonstrated the ease of genetic manipulation of LUHMES cells by CRISPR/Cas9 approaches, generated seven mutant MECP2 LUHMES cell lines and explored the potential of protein therapy as a therapeutic approach for RTT. The LUHMES cell line proves to be extremely easy to handle and robust and has yielded novel molecular insights into the function of MeCP2 in human neurons. In particular, MeCP2-null cells show a striking relationship between the level of gene body methylation and the extent of transcriptional upregulation when compared to wild-type neurons. In contrast neurons that express a form of MeCP2 that can bind to DNA but cannot recruit a transcriptional corepressor complex (the R306C mutant) do not exhibit substantial gene expression alterations, yet do display a consistent decrease in total RNA amount. This decrease in total RNA is recapitulated in MeCP2-null LUHMES-derived neurons and in brain regions from MeCP2-R306C mice. The requirement for functional DNA binding for normal gene-body methylation dependent gene repression is demonstrated by assessing LUHMES cells that overexpress MeCP2-R111G, a protein that cannot bind to DNA. Furthermore, overexpression of the MeCP2-R306C protein highlights the importance of NCoR binding for normal gene repression, but also demonstrates that MeCP2-R306C protein retains some gene repression activity. Thinking more broadly, this cell line also has applications as a model system for a variety of other neurological disorders; as a simplified model system to elucidate molecular and neurological phenotypes, and as a relevant human system that can be cultured in a high-throughput manner for testing therapeutic strategies.

The haematopoietic phosphatase PTPN22 is a key regulator in balancing immune responses between self-reactivity and tolerance. PTPN22 downregulates T cell signaling and harbors the non-HLA genetic variation most strongly associated with autoimmune disease in humans, the single nucleotide polymorphism R620W. The effect of this mutation is currently controversial due to confounding results in mouse and human models. The polymorphism is linked to increased susceptibility to autoimmunity in both human and mouse models, although the latter does depend on genetic background. However, mouse data clearly shows that the polymorphism has a loss-of-function effect on T cell signalling, whereas studies in human models largely demonstrate a gain-of-function effect for R620W. A confounding issue in human studies is that they depend on comparison of T cells from distinct individuals, on protein over-expression, or on RNA interference, techniques for which it is difficult to control for genetic and environmental variables, changes in stoichiometry, and off-target effects or incomplete knockdown, respectively. We aimed to create isogenic human cell lines with mutations in PTPN22 at the genomic level to alleviate the complications inherent in analysing human data. In addition to autoimmune pathogenesis, we are interested in the role of PTPN22 in a cancer setting. Because PTPN22 has a strong suppressive effect on T cell responses to weak affinity antigen, which encompass most tumour antigens, we postulated that knocking out PTPN22 may better enable T cells to kill tumour cells. Furthermore, we have shown that PTPN22 knockout (KO) leads to increased IL-2 expression in mouse T cells, and that this effect is protective against TGF-β mediated suppression, a common driver of T cell inhibition in the tumour microenvironment. T cell transfer experiments in mice showed that PTPN22 KO T cells are indeed more effective at reducing tumour size. Based on these findings, we aim to determine whether PTPN22 KO in human cells confers a similar effect on signaling. To investigate the effects of PTPN22 KO on human T cell signaling, we used CRISPR gene-editing to target PTPN22 in a Jurkat cell line. By combining this technique with lentiviral transduction of a specific T cell receptor, we generated human cell lines which are genetically identical, save for specific alterations to PTPN22, and which can be stimulated with strong or weak cognate antigen. We found that PTPN22 KO Jurkat cells develop an enhanced activation phenotype upon stimulation, including increased IL-2 expression. Additionally, PTPN22 KO Jurkat cells show enhanced Erk signalling following stimulation with weak affinity antigen, but this difference is lost as stimulus strength increases. CRISPR technology has presented the opportunity to create novel models of PTPN22 signalling in the context of human T cell lines. The data from these lines suggests that, unlike the R620W mutation, complete loss of PTPN22 has a comparable effect in human and mouse T cells. In conjunction with our previous findings, these results suggest that knocking out PTPN22 may lead to signalling alterations that improve adoptive T cell cancer therapy.

Includes nine pages of amendments. / Bibliography: leaves 140-211. / 211, [200] leaves, [12] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to determine the reason for the lack of ability of many myeloid leukaemic cell populations to stimulate allogeneic lymphocytes in mixed leucocyte culture (MLC), with a view to manipulating the immunogenicity of these cells for therapeutic purposes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995

Helicobacter pylori infects and persistently colonizes the stomach, which results in gastritis and in some individuals peptic ulcer disease or gastric cancer. Adherence of H. pylori to the epithelium is an important factor for development of disease. Attachment is mediated by the adhesins BabA and SabA that binds the ABO/Leb blood group antigens and sialylated glycoconjugates respectively.  High-affinity attachment could be anticipated to be of disadvantage for H. pylori because epithelial cells have a fast turnover rate and the dislocated and shed epithelial cells would carry attached bacteria to the acidic gastric juice in the lumen. However, here we describe that H. pylori manage to adapt to this innate clearance mechanism by unique acid regulatory binding properties of its adhesins. We propose that pH regulated binding properties enable bacteria to detachment from host cells for chemotactic guided motility and successful return to the more neutral epithelium for a fresh restart of the infectious cycle. By comparison of BabA from different stomach loci we identified amino acid key position for acid regulated binding activity. Previous studies found lower prevalence of Leb-binding among H. pylori isolates from southern Europe compared to Sweden. Here we tested if the reduced prevalence of Leb-binding could be explained by a novel binding mode; in among Spanish strains, we identified S812 that demonstrates preference for multivalent binding to ABO antigens in glycolipids; we found that 812 BabA had drifted in its preferred binding epitope away from the consensus a1,2fucosylation and towards the blood group A and B derivatives. Such epitope drift might in particular optimize binding to ABO antigens in densely packed lipid rafts. In parallel, we studied the influence of BabA for disease progression by an inventory of gastric biopsies. BabA correlated both with the oncoprotein CagA, the VacAs1 toxin and, in addition, to severe disease progression. We further correlate BabA expression with positive secretor phenotype and stronger adhesion of H. pylori in vitro. For functional adherence studies in vitro, we constructed a recombinant Leb-expressing cell lineage that supports BabA mediated H. pylori attachment.