Dissertationen zum Thema „Hesci“

Diabetes is a chronic disorder of the pancreas, where a decline in the insulin-producing β-cell population disrupts metabolic homeostasis. Pancreatic transplantation has shown to be effective in circumventing the problem of β-cell insufficiency. However, availability of donor islets remains an obstacle. Although progressive differentiation of embryonic stem cells (ESCs) to pancreatic β-cells is a solution, current protocols are wrought with inefficiencies. It is obvious that to realize ESC differentiation for therapy many steps need to be optimized, and this study describes improvement of Pdx1+pancreatic progenitor derivation, a critical determinant of pancreatic fate. The compounds melatonin and sPDZD2 have been suggested to act through the Protein Kinase A (PKA) pathway to exert transcriptional effects, and in particular sPDZD2 stimulates the expression of pancreatic genes in INS-1E rat pancreatic cells. This led to the hypothesis that the PKA-targeting characteristics of said molecules could be exploited for pancreatic specification through post-translational activation ofPdx1. hESCs were first induced to form definitive endoderm before treatment with melatonin and sPDZD2. Pdx1 expression induced by these molecules was then compared with levels triggered by known pancreatic progenitor inducer Indolactam V (ILV). A secondary objective of this study was to assess the endoderm induction potential of small molecules in hESCs, which claim to be potentially useful in differentiation. In this research, I show that small molecules are noticeably more challenging to use in the hESC context. Between the TGF-β pathwayactivatorsIDE-1 and 2, the latter is more potent at inducing endoderm formation, though it does not surpass the capabilities of Stauprimide, a molecule originally thought to only serve a priming purpose in mESCs.IDE-2 and Stauprimide consistently perform better than Activin A, the near universal factor for endoderm induction. Possible synergy between IDE-2 and Stauprimide was explored, but their combination appears detrimental to Sox17expression. Subsequent pancreatic differentiation was also inefficient, and my results affirm the immaturity of chemically-induced endoderm by contrasting with mainstream means of endoderm induction; levels of endoderm marker expression between the two methods are millions of folds apart. This work exposes the risks of using small molecules, and they necessitate proper characterization before being adopted for differentiation. Most favorably, both sPDZD2 and melatonin were able to trigger Pdx1 expression in STEMDiffTm derived definitive endoderm; 10 and 30folds respectively, comparable to the known Pdx1 inducer ILV (25 folds). I also reveal concentration-mediated differentiation and proliferative purposes of ILV and sPDZD2, which are highly reminiscent of the signaling mechanisms involved during pancreatic development. Preliminary quantification of Pdx1+ cells suggest that high concentrations of ILV and sPDZD2 favor self-renewal of Pdx1+ progenitors, whilst lower doses elevate Pdx1 expression. Demonstration of Pdx1 at both gene and protein expression levels was encouraging, but it remains uncertain if melatonin and sPDZD2 manipulate PKA signaling to exert Pdx1 promoting effects. My work supports the use of melatonin as a candidate for pancreatic differentiation, and suggests involvement of sPDZD2 in deriving and expanding progenitors during pancreatic organogenesis.
published_or_final_version
Biochemistry
Master
Master of Philosophy

The thesis examines the processes as they occur in the actor ́s mind during work on the character while rehearsing and acting. It also describes extensively the steps prior to the rehearsal process and acting from the actor ́s point of view regarding the actor ́s mental states. Additionally, the focus is on various approaches to character building during rehearsals and on various wals of working with the text. In her analyses, the authoress is aware of the specific differences of the theatrical genres. The thesis is based largely on her experience.

This study examined the value of the HESI Admission Assessment in predicting student success. Associate degree (N ≤ 68) and baccalaureate (N ≤ 69) nursing students took the HESI Admission Assessment after acceptance into the nursing programs for the purpose of identifying their academic weaknesses and focusing their remediation efforts. Findings indicated that the HESI Admission Assessment was a valid predictor of students' academic ability to succeed in the nursing programs. In the associate degree nursing program, HESI Admission Assessment scores were significantly positively correlated with 88.89% of all nursing course grades in the program and 100% of the beginning-level course grades. In the baccalaureate nursing program, HESI Admission Assessment scores were significantly positively correlated with 50.00% of all nursing course grades in the program and 80.00% of beginning-level course grades. Furthermore, associate degree nursing students who completed the program had significantly higher HESI Admission Assessment scores than those who did not complete the program.

Mon travail a consisté à caractériser la monocytopoïèse dérivée d'hESC et à confirmer in-vitro que les macrophages obtenus partageaient les mêmes fonctions homéostatiques que ceux dérivés de foetus. J'ai ensuite dérivé des lignées d'iPSC à partir de cellules CD34+ d'un sujet sain et muté JAK2V617F. Ma première étude a consisté à comparer les macrophages issus de la différenciation d'hESC à ceux issus d'iPSC contrôle. Cette partie de mon travail a permis de montrer que les macrophages issues d'IPSC sont très proches de ceux dérivées de hESC mais présentent des différences significatives dans la sécrétion de certaines molécules de la réponse inflammatoire à l'IFN-y et au LPS. Enfm nous avons souhaité étudier grâce à la technologie des iPSC l'implication de la voie JAK/STAT dans le phénotype fonctionnel des macrophages embryonnaires et dans leur défaut de réponse à l'IFN-7 et au LPS. Les résultats préliminaires ne montrent ni de différences dans la polarisation des monocytes/ macrophages JAK2V617F, ni d'altération dans l'activation de STAT1 via l'IFN-7. Les différences ontogéniques sont donc liées soit à des modifications épigénétiques dans les gènes impliqués dans l'inflammation ou dans les partenaires de STAT1. Un travail encore plus préliminaire a été d'explorer les éventuelles indépendances au bFGF des iPS( pluripotentes. En effet un travail de Griffiths et al a montré que les mESC JAK2V617F pouvaient mainteni leur phénotype pluripotent en l'absence de LIF via son action nucléaire indirecte sur le promoteur de Nanog Les résultats préliminaires montrent que les lignées d'IPSC JAK2V617F humaines pouvaient maintenir li phénotype pluripotent et cela en l'absence de bFGF
My work was focused on the monocyte and macrophage lineages. We have shown that Monocyte/macrophage derived from ES cells are cells extremely specialized in tissue remodeling, pro-angiogenesis and immune suppression but with low inflammatory potential. I have investigated the characteristics of monocytes/macrophages from iPS. Those monocyte/macrophage were quite similar to hESC derived, but exhibited more inflammatory potential suggesting some incomplete reprogrammation during derivation of IPS. We hypothesize that the decrease or absence of inflammatory potential of the monocyte/macrophages could be related to a decrease activation of the JAK2/STAT pathway induced by IFN-y and GM-CSF, two cytokines implicated in MI polarization. In this purpose we derive iPS from patient harboring the JAK2V617F mutation, a gain of fonction mutation associated with myéloproliférative neoplasm. In preliminary results, no significant differences were observed in the polarization of JAK2V617F or JAK2WT monocyte/Macrophages derived from IPS. Furthermore we found that IFN-y was capable to normally induce STAT1 activation in these cells suggesting that the blockage in inflammatory response is downstream STAT1 and may be related to epigenetic regulation of inflammatory gens. Finally I have obtained preliminary results showing that JAK2V617F may induce independence to bFGF of the pluripotent iPSC, a result very similar to those reported by Griffiths and al on JAK2V617F mESC who could maintain their pluripotent phenotype without addition of LIF. This was not related to the induction of the canonical STAT pathway but to an effect of nuclear JAK2 on the epigenetic regulation of Nanog

In my master thesis, I am trying to name the origins and signals of an actor`s fear. I have came from my own existing stage experiences previously and whilst studying classical acting at the Dramatic Faculty of the Academy of Performing Arts in Prague, and also from my first experiences with differing professional groups from theatres from around the Czech Republic. My thesis is supported by professional literature, with which one I have read during my studies at the Academy, and it is inspired by talks and by consultation with people who are active in an artistic field. In my work I reflect my mental prosesses, states and blocks, not only during stage creations, but also in my personal life. I think that a fear can rule an actor and cause stage fright. This is why I have decided to choose this subject for my thesis.

This magister thesis is about the task of contemporary performer in mime theater. The thesis deals with a question of authorship and physical being of the performer on the stage. The thesis is based on analysis of graduation performence of Jindřiška Křivánková titled The hunters and on the basis of this analysis entitle principals of authorial theater and contemporary acting.

This thesis deals with theme The actor and the monologue. After defining the concept of monologue, there is an excursion into history and the development of the monologue. There are given personal experiences of working on monologues during the studies and talks about transformation of the ideas about monologue. In the end there is also definition of the concept of monodrama and experiences with unfinished work on monodrama by Ivan Vyrypajev called July.

Work in our lab has resulted in the development of a novel approach to creating a more developmentally plastic human dermal fibroblast (hDF) phenotype that allows for the study of molecular mechanisms involved in cell-fate conversion. Culturing hDF under defined culture conditions (5% O2 and supplementation with fibroblast growth factor FGF2) induces induced the regeneration competent (iRC) phenotype that is characterized by stem cell gene expression, and increased life-span in vitro. The work presented in this thesis further characterizes the system, and describes an overall shift in extracellular matrix and adhesion molecules in human dermal fibroblasts (hDF) undergoing the transition to a more developmentally plastic phenotype (iRC). This work suggests that we create the initiation phase of Mesenchymal-to-Epithelial Transition (MET) during conversion to the iRC phenotype. This transition is marked by loss of integrin alpha-11 (α11) and its binding partner Collagen-I (COL-I). Moreover, we describe the mechanism for the down-regulation of α11 that is mediated by FGF2 activation of ERK1/2 through systematic investigation of several potential molecular mechanisms. The body of work presented here shows that the ERK 1/2 mediated down-regulation of α11 is independent of activation of TGF-β1-mediated regulation of α11. In addition to down-regulation of α11, an overall shift in the transcript levels of other adhesion molecules is observed, which demonstrates that iRC are most likely transitioning their attachment to a laminin and fibronectin-based matrix. These results suggest that iRC may be producing a more “pro-regenerative matrixâ€�. We hypothesize that the changes in integrin expression profile and interaction with ECM serve as a feedback loop during the iRC phenotype shift. Our findings suggest that this “pro-regenerativeâ€� shift in attachment of iRC as well as the ERK 1/2 mediated down-regulation of α11 could be exploited in wound healing biology and fibrosis research. Manipulation of the dynamic relationship between TGF-β1 and FGF2 has the potential to reduce scar deposition. Further identification of molecular mechanisms controlling this phenotype conversion will allow development of strategies for in situ manipulation of wound healing outcomes.

The endometrium undergoes recurrent cycles of dynamic remodelling, involving breakdown and scarless repair, proliferation and differentiation, including decidualisation of the stroma, during the menstrual cycle. Extensive studies have characterised how the steroid hormones oestrogen and progesterone acting via their nuclear receptors coordinate these remarkable changes. Although a few previous studies have postulated a role for androgens the impact of androgens on endometrial function remains understudied. The studies described in this thesis aimed to 1) identify cellular processes, pathways and networks regulated by androgens in human androgen receptor-positive endometrial stromal cells (hESCs), 2) investigate the potential for regulation and determine the regulation of putative dihydrotestosterone (DHT)-regulated gene expression by androgen in hESCs, 3) investigate the expression and regulation of putative androgen-regulated genes in the human endometrium across the menstrual cycle and in early pregnancy and 4) explore the role of androgens in modulating metformin-induced gene expression associated with decidualisation of hESCs. Analysis of data from a whole genome array conducted previously in the laboratory using primary hESCs treated with DHT for 2 or 8 hours identified time dependant putative androgen-regulated mRNAs (34 and 268 genes, respectively). Thereafter, all work was completed by the author. Gene ontology and functional based bioinformatic analyses of the putative androgen-regulated gene sets revealed potential androgen regulation of a variety of cell processes, pathways and networks including those associated with gene transcription, signal transduction pathways (such as phosphatidylinositol, oestrogen receptor alpha (ERα) and Wnt signalling), cancer pathways, metabolism, cell cycle, development, apoptosis/survival. In addition, various transcription factors (e.g. AR, c-Myc, SP1, ERα, p53, E2F1, RUNX2, CREB1 and STAT3) were associated with androgen regulation in hESCs. Consensus androgen receptor binding sites were identified in the promoter sequences of 18 genes by transcription factor binding site sequence analysis. Direct DHT regulation of ten of 15 of these genes was validated in endometrial stromal cells using qRTPCR. Of these genes, RGS2, SIK1, and SNCAIP mRNAs were confirmed as DHT-regulated in hESCs by use of an AR inhibitor (flutamide) and in addition, were not found to be regulated by oestradiol. Discovery bioinformatics predicted these genes may interact in a gene network involving AR and the cAMP transduction pathway. Expression of the 15 putative androgen-regulated genes was confirmed by qRTPCR in intact human endometrial tissue (13 novel) and 9 of these genes were regulated in association with decidualisation i.e. either in the secretory phase, the time at which decidualisation begins and/or in first trimester decidua. Protein expression of RGS2, SIK1 and Synphilin-1 (encoded by SNCAIP) was confirmed by immunohistochemistry in endometrial tissues and protein expression also appeared greater in decidua. Regulation of putative androgen-regulated gene expression by decidualisation was confirmed in 4 out of 8 genes by employing a model of reduced in vivo decidualisation i.e. decidua from ectopic pregnancies. Regulation of 5 out of 7 genes was confirmed in decidualised hESCs (RGS2, SIK1, SLC6A6, SNCAIP and AXIN2) but expression of these genes was not altered by DHT inclusion during decidualisation. Finally, only a high metformin concentration enhanced hESC decidualisation and putative androgen-regulated gene expression (4 genes) in decidualised hESCs. In comparison, in the presence of DHT, a lower clinically relevant metformin concentration (100μM) did enhance decidualisation marker expression but did not alter expression of putative androgen-regulated genes. In summary, these studies have revealed new insights into androgen action in the human endometrium. Studies in hESCs 1) predicted the pathways and interacting transcription factor regulatory networks that may be androgen-dependent in this cell type, these were associated with cell differentiation, apoptosis and proliferation, 2) identified novel putative androgen-regulated genes expressed in hESCs and in endometrial tissues, 3) showed putative androgen-regulated genes are regulated by DHT (possibly via AR) in endometrial stromal cells, some of which are also regulated in association with decidualisation and 4) showed that androgens may enhance decidualisation during exposure to the commonly used drug metformin. Collectively, these new findings support a physiological role for androgens in endometrial function and provide a series of new avenues for further studies of the regulation of differentiation and proliferation.

Dissertation presented to obtain a Master degree in Biotechnology at the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia
Human embryonic stem cells (hESCs) are known by their ability to either self-renewal and differentiate into any adult cell type. These properties confer to hESCs a huge applicability for cell therapy, tissue engineering and drug screening. However, successful implementation of hESCs-based technologies requires the production of large numbers of well characterized cells and their efficient long-term storage. In this study, alginate microencapsulation technology was used in order to develop an efficient, scalable and integrated 3D culture system for expansion and cryopreservation of pluripotent hESCs. Three strategies were outlined: microencapsulation of hESCs as single cells, cell aggregates and cells immobilized on microcarriers. Encapsulation of hESCs immobilized on microcarriers was the best strategy to expand and cryopreserve pluripotent hESCs. The culture of encapsulated hESCs-microcarriers in spinner vessels assured an approximately 20-fold increase in cell concentration. Moreover, this strategy improved twice cell survival after cryopreservation by a slow-freezing rate procedure, comparatively with non-encapsulated culture. Microencapsulation also protected hESC aggregates from damage caused by stirring, allowed the control of aggregates size and the maintenance of cells pluripotency for two weeks. This work demonstrates that microencapsulation technology is a powerful tool to enhance growth and post-thawing recovery of pluripotent hESCs. The 3D culture systems developed herein represent a promising vehicle to assist the transition of hESCs to the clinical and industrial fields.
This work was performed in the scope of the project - Integrated strategy for expansion, neuronal differentiation and cryopreservation of human embryonic stem cells (PTDC/BIO/72755/2006) funded by FCT (Fundação para a Ciência e Tecnologia)

Differentiation and cell cycle regulation in stem cell have a key function for embryonic development, organ homeostasis and tissue repair. Recent results have shown that these two mechanisms are intrinsically connected. Indeed, cell cycle machinery directly controls maintenance of pluripotency and initiation of differentiation. More precisely, the cell cycle regulator Cyclin D appears to control the transcriptional activity of Activin/Nodal signalling during progression of the cell cycle in human Embryonic Stem Cells (hESCs). As a consequence, hESCs can only differentiate into endoderm in the Early G1 phase when Cyclin Ds are expressed at low levels. These results show the mechanisms by which the cell cycle defines differentiation propensity of stem cells. However, these observations also imply the existence of interplays coordinating extra cellular signalling pathways with the epigenetic state, chromatin structure and transcriptional networks during cell cycle progression and these mechanisms remain to be fully uncovered. Here, I have utilised the FUCCI reporter system combined with ATAC-Seq to analyse chromatin dynamics during cell cycle progression in hESCs. Furthermore, I performed ChIP-Seq analyses to define the genomic location of transcriptional regulators during cell cycle progression as well as RNA-Seq to confirm variation in gene expression pattern. Integration of these data shows that the chromatin status in hESCs is highly dynamic and the core pluripotency transcription factors and epigenetic modifiers change genomic location during cell cycle progression. I also showed that hESCs in the Late G1 phase accumulate transcripts that are important for differentiation and development; therefore, indicating this phase represents a unique portion of the cell cycle for cell fate decisions. Taken together, these results uncover that transcriptional networks are unexpectedly dynamic during the progression of cell cycle in stem cells. I hypothesise that these modifications are necessary to prime hESCs for different cell fate choices allowing a diversity of differentiation that is otherwise impossible. Overall these mechanisms underline the need to study transcriptional and epigenetic mechanisms in the dynamic context of the cell cycle and have major implications for adult tissue homeostasis and disease.

I would like to understand the importance of an actor in a movie. I choosed Serge Reggiani, a french actor, who never became a star number one. Reggiani went through a lot of important movies in principal or small roles. What it really means acting in a movie, what brings freedom to an actor in front of a camera and is one actor able to play different characters in every other movie. A diversity and length of his filmography, different directing styles he went through will help me to concretize attitude of an actor and perception of his „different“ characters by audience. I would like to bring up some acting metodes in cinematography in my work too.

This Master's thesis addresses the process of staging musicals and operettas within the Czech theatre?s environment. As an example, the author chose five different titles for comparison and description of the process of preparation of this specific kind of musical theatre focusing on various options for theatre companies, discussing the demands and difficulties of assembling teams of artistic collaborators and the creative process itself during rehearsals. Processes concerned with the first grasp of a title as a dramatic text, through the spatial solutions of a dramatic situation in terms of stage space in a theatre, up to the director-actor collaboration, are documented by authentic findings, which are compared with notes of directors, who significantly influenced the author's view. The text also considers the specific demands of this kind of theatre on the actor (actress) and his (her) education and training

Human embryonic stem cells (hESCs) can provide a unique approach for novel tissue engineering applications. Previous groups have shown that hESCs can differentiate into specialized cell types through the generation of human embryoid bodies (hEBs). These multi-cellular constructs are then subjected to suspension culture for several weeks. Traditional hESC differentiation techniques have yielded non-homogeneous EBs derived in standard static cultures providing an inefficient platform for cellular viability and embryonic modeling. Here, our study aimed at systematically comparing the formation, growth, and differentiation capabilities of hESC-derived hEBs in dynamic and static suspension cultures. We used a continuous flow perfusion slow turning lateral vessel, STLV, system (Synthecon) to model after an in vivo environment. This study is in part of a larger study investigating the role of HOXB5 in the human endothelial differentiation pathway. Embryoid bodies were created by hanging drops and then subjected to static or dynamic culture for 10 days. Cells were harvested and a simple Alkaline Phosphatase assay was used to determine if the system was viable for propagating hEB. We show that the STLV system is viable for our future studies and this system more efficient at maintaining hEBs.

Ce travail étudie les mécanismes physiopathologiques de deux thrombopénies héréditaires (TH). Premièrement on identifie des anomalies génétiques causant une nouvelle TH de transmission autosomique récessive, associée à une tendance au saignement et un défaut de réorganisation du cytosquelette de formation des proplaquettes (PPT) et d'activation des plaquettes. On identifie par séquençage exomique une mutation homozygote du gène PRKACG qui entraîne une perte de fonction. On montre que la mutation est associé à un défaut important dans la formation des PPT ainsi qu'à une diminution de filamin A dans les mégacaryocytes La surexpression de la forme WT de PRKACG restaure le phénotype in vitro observé chez les patients confirmant. La deuxième TH étudiée est FPD/AML qui est causée par des mutations dans le gène RUNX1. Certaines mutations prédisposent aussi aux leucémies et pour comprendre comment on a établi des lignées de cellules souches pluripotentes induites (iPSC) à partir de 2 familles porteurs des mutations germinales. La première porte une mutation R174Q qui agit comme un dominant-négative (DN) et qui cause TH et prédisposition aux leucémies ; la deuxième porte une délétion monoallelique du gène induisant une haploinsuffisance qui cause seulement une TH. L'étude de l'hématopoïèse à partir de ces iPSC a montré un défaut profond dans l'érythropoïèse et dans la megakaryopoiesis ainsi qu'une forte dérégulation des gènes cible de RUNX1. Seulement les progéniteurs issus des iPSC DN montre une augmentation des granulo/mono-cytes, un phénotype reproduit par le knockdown de 80% de RUNX1 dans les cellules souches embryonnaires H9, ainsi qu'une augmentation dans l'instabilité génomique
In this work we study the pathogenesis in two distinct inherited thrombocytopenias (IT). We started by the identification of the genetic abnormality causing a new IT. This IT is transmitted in an autosomal recessive manner and is associated with severe bleeding phenotype, a defect in the cytoskeleton reorganization, decreased proplatelet formation, and deficiency in platelet activation. Using exome sequencing, we identified a new homozygous mutation in the PRKACG gene. This gene encodes a γ-catalytic sub-unit of the PKA, and the mutation leads to loss of function. We show that the PRKACG mutation is associated with a marked defect in proplatelet formation and a low level in filamin A in megakaryocytes. We confirm that the thrombocytopenia is due to mutation in the PRKACG gene since the overexpression of WT PRKACG reverses the phenotype observed in patients in vitro. We also studied FPD/AML — IT caused by RUNX1 mutations. Some mutations also predispose to leukemia, and to understand how, we generated induced pluripotent stem cells (iPSCs) from 2 pedigrees with germline mutations. One carries a R1 74Q mutation, which acts as a dominant-negative (DN) and is associated with thrombocytopenia and leukemia; the second carries a monoallelic gene deletion inducing a haploinsufficiency, which causes only thrombocytopenia. The study of hematopoiesis from these iPSC clones demonstrated profound defects in erythropoiesis and megakaryopoiesis and deregulated expression of RUNX1 targets. Only progenitors from DN iPSC clones showed an increased amount of granulo/mono-cytes, a phenotype reproduced by an 80% RUNX1 knockdown in the H9 human embryonic stem cell line, and a genomic instability

Stem cell pluripotency and self-renewal are two important attributes of human embryonic stem cells which have led to enhanced interest in stem cell research. Understanding the mechanisms that underlie the regulation and maintenance of these properties is imperative to the clinical application of stem cells. Pluripotency and self-renewal are regulated by different genes, transcription factors and other co-factors such as FoxD3 and Klf4. Oct4, Nanog and Sox2 are central to the stem cell regulatory circuitry. They form interactions with co-factors to promote cell proliferation and inhibit differentiation by negatively regulating differentiation markers. However, there are other novel pluripotency associated factors yet to be studied. In this study, bioinformatics and functional analyses were employed to identify a potential pluripotency gene called YY1AP1 from our lab's pre-existing microarray data. YY1AP1, a transcription regulatory gene, showed consistent down-regulation with induced cell differentiation. It was further investigated. First, its co-localization with Oct4 in both hESCs and iPSCs was confirmed by immunofluorescence staining. Knockdown experiments were then performed on this gene to investigate effects of knocking it down on gene expression in hESCs. Knocked-down cells were characterized for markers of pluripotency and differentiation at the transcript level. Results showed a down-regulation of pluripotency genes with no specific promotion of any of the germ layer markers. Gene expression at the protein level in knocked down cells was then assessed for YY1AP1, and its binding partner YY1, and pluripotency markers. Results showed that proteins of YY1AP1, YY1, Oct4, Nanog and CTCF were down regulated while the tumour suppressor gene protein, p53, was up-regulated in YY1AP1 deficient stem cells. Protein to protein interaction studies showed that YY1AP1, YY1, Nanog and CTCF proteins directly interacted with each other. Differentiation of YY1AP1deficient cells into EBs led to an almost complete shutdown of all gene expression, an indication that the cells did not form 'real' EBs. Differentiation of YY1AP1 ablated cells did not support any lineage promotion either. These results suggest a potentially new role for YY1AP1 in proliferation and self-renewal of stem cells through its possible direct binding to CTCF or its indirect binding to CTCF in complex with YY1.

Huntington’s Disease (HD) is a neurodegenerative disorder cause by a dominant CAG triplet repeat expansion (poly-glutamine(Q) in the protein) in the first exon of the Huntingtin gene (HTT). In the healthy population, the number of CAG repeats range between 9 and 35, while an expansion above 36 CAG repeats causes the manifestation of the pathology later in life. Numerous studies indicate that the pathological CAG length causes the poly-Q expanded protein to acquire a toxic function which ultimately kills the neurons1–10. Other studies show that several disfunctions associated with the presence of the mutant protein can be phenocopied in cells and mice deleted of the healthy gene, suggesting that a loss of function mechanism may also contributes to HD11–13. Thus the disease seems to be the result of a two-component pathological mechanism; first a loss-of-function (LOF) due to reduced normal HTT physiological activity, and second a gain-of-function (GOF) due to mutant HTT toxic effects14. Being able to discriminate between LOF and GOF phenotypes is especially relevant when considering the ongoing gene silencing clinical studies aiming at reducing HTT level either in an allele-specific or non-allele-specific manner15–17. On top of that, transcriptional alterations have been reported both in HD patients and HD mouse models18, and several evidence have linked HTT to gene expression1,19–27. However, these data refer to situations in which a transcriptional balance has already been established following the genetic perturbation. No data is available regarding the mechanisms acutely implemented by the cell immediately after perturbation to re-establish transcriptional balance in response to those changes. For these reasons, in this project we aimed at generating a human embryonic stem cell (hESC) HD model in which normal or mutant HTT can be rapidly and efficiently removed upon exposure to a small molecule. This is made possible by a degradation tag (dTAG)28 that, when fused to HTT, induces a proteosome-mediated degradation upon exposure to a cell permeable ligand. Accordingly, the first part of the project consists in the generation of two dTAG-hESC-HD lines in which either the normal or the mutant allele were targeted by Cas9-assisted genome editing followed by the assessment of the efficiency and degradation kinetic of tagged HTT in both cell lines. These experiments revealed complete tagged HTT loss between one and two hours of treatment with a ligand concentration of 10-7 M. Moreover, no difference was observed when comparing degradation kinetics of normal and mutant HTT in self-renewing dTAG-hESC lines. We aim to use these lines to study the immediate transcriptomic changes and the potential compensatory mechanisms established by the cells in response to normal or mutant HTT protein depletion. Therefore, a first RNA-seq experiment was performed to investigate over time transcriptional changes during dTAG-HTT degradation. This experiment revealed no substantial transcriptomic changes between normal and the HTT-depleted state of self-renewing dTAG-hESC HD lines. We are now looking into the transcriptional changes driven by HTT degradation in hESC in vitro derived neurons that can provide useful information on the biosafety of the ongoing HTT-lowering approaches for HD treatment.

The thesis describes my personal experience which I gained during my studies at the Academy of Performing Arts (DAMU), rehearsals at the Theatre Studio Disk as well as experience gained out of school. This experience is illustrated with several examples of practical work which I participated in. Specially is described the interaction with the director, my theatrical partners, theatre-goers and with my own during the process of rehearsals.

This thesis is about acting in theater or in front of camera. At the beginning it is focused to these directions individually and later summarizes their differences, which include: what function the audience has; what options of movement the performer has; what style direction the actor uses; what the difference of collaborations between actors on the stage or in front of the camera have.It talks about the different approach of directors and self-expressions of actors and analyzes the different effects that each actor during their performance. It doesn’t only talk about the differences, but also includes the inter-connections of both styles. The next part is focused on creating a character and comparison of current and passed actors that were acting in theater as well as in movies. The conclusion is about my personal acting experience in film and in theater. It evaluates my work at the school theater DISK and also asses the transition to my advanced work.

Primary human hepatocytes are a scare resource with limited lifespan and variable function which diminishes with time in culture. As a consequence, their use in tissue modelling and therapy is restricted. Human embryonic stem cells (hESC) could provide a stable source of human tissue due to their self-renewal properties and their ability to give rise to all the cell types of the human body. Therefore, hESC have the potential to provide an unlimited supply of hepatocytes. To date, the use of hESCs-derived somatic cells is limited due to the undefined, variable and xeno-containing microenvironment that influences the cell performance and life span, limiting scale-up and downstream application. Therefore, the development of highly defined cell based systems is required if the true potential of stem cell derived hepatocytes is to be realised. In order to replace the use of animal derived culture substrates to differentiate and maintain hESCs-derived hepatocytes, an interdisciplinary approach was employed to define synthetic materials, which maintain hepatocyte-like cell phenotype in culture. A simple polyurethane, PU134, was identified which improved hepatocyte performance and stability when compared to biological matrices. Moreover, the synthetic polymer was amenable to scale up and demonstrated batch-to-batch consistency. I subsequently used the synthetic polymer surface to probe the underlying biology, identifying key modulators of hepatocyte-like cell phenotype. This resulted in the identification of a novel genetic signature, MMP13, CTNND2 and THBS2, which was associated with stable hepatocyte performance. Importantly, those findings could be translated to two hESC lines derived at GMP. In conclusion, hepatocyte differentiation of pluripotent stem cells requires a defined microenvironment. The novel gene signature identified in this study represents an example of how to deliver stable hESCs-derived hepatocytes.

In my master thesis I focus on psychosomatic, internal and external, actors assumptions, which are an essential part of acting talent. I deal with the question of to what extent is an anctor influenced by the nature of man, his qualities and character. The work is examining the relationship between the body and the psyche, especially its impact on creating a dramatic character during the rehearsing process and the overall speaking and physical stage of negotiations. I am naming not only my own acting assumptions, but also the problems that accompanied me during the study, whether cloistered and in graduate roles in the theater Disk . I am referring to the actor's bad habits, risks of relations between acting partners and creative team as well as the necessity of pursuing the profession responsibly and artistically, realize of my strengths and weaknesses and targeted work to eliminate them.

The thesis is focused on basic understanding of the actor as an acting component of the theatre piece and follows his work and creation during the life theatre performace as well as during the rehearsing process (with description of general acting technics).

The purpose of this study was to evaluate the relationship of pre-entrance factors and the success of students in an Associate of Applied Science (A.A.S.) degree nursing program at a community college in East Tennessee. The criterion variable was success in the nursing program. Success was defined as academic success in all nursing courses and completion of the nursing program to graduation. The predictor variables of pre-entrance factors were gender, age, Health Education Systems, Incorporated (HESI) A2 scores, Pell Grant eligibility, pre-nursing GPA, and prior licensure. The data for this non-experimental secondary analysis were derived from the electronic database in the community college Banner system. The population of the study consisted of all students accepted into the A.A.S. Nursing Program at a selected community college for academic years beginning 2013-14, 2014-15, and 2015-16. The population of the study was approximately 300 students. Analyses of the data were completed using independent samples t-test and chi-square cross tabs. Findings revealed that the mean HESI A2 scores were higher in those students that successfully completed the Nursing program than those that did not complete the program. Findings revealed a statistical significance between gender and program completion with females more likely to successfully complete the nursing program than male students. The factors that had no significant relationship to successful completion were age, high school GPA, Pre-nursing GPA and holding prior licensure. Findings also revealed students who are Pell eligible were not significantly more likely to complete the nursing program than those students who were not Pell eligible.

High-stakes licensure or certification examinations are required for many health professions disciplines to ensure safe entry-level practice. Accrediting agencies set a benchmark for graduates' first-time licensure or certification success as a measure of program effectiveness. Failures of graduates on licensure or certification examinations may directly affect the school's recruitment and retention of qualified students and faculty, as well as institutional financial viability. A health science university has added Health Education System, Inc. (HESI) standardized examinations using computer adaptive testing into the family nurse practitioner (FNP) master's program to support certification success, although research on these advanced practice examinations as related to certification outcomes was lacking. Guided by classical test theory, this study was an investigation of whether a relationship existed between students' performance on 4 HESI standardized examinations (Advanced Pathophysiology, Advanced Pharmacotherapeutics, Advanced Health Assessment, and the APRN/FNP Exit exam) and first-time FNP certification success. Binary logistic regression analysis of data from 117 students who graduated between 2013-2016 indicated that none of the 4 standardized HESI examinations significantly predicted FNP certification success, perhaps due to the examinations not carrying any evaluative weight within the program. The results of this project study may be used to promote positive social change by providing a means to improve first-time certification success and increasing the availability of primary care providers in the role of FNP.

Systematic, genome-wide interrogations have identified hundreds of genes, including several transcription factors, which have expression patterns tightly correlated with ES cell differentiation. OCT4, SOX2 and NANOG constitute a triad of transcription factors, identified as crucial for the maintenance of ES cells self-renewal and pluripotent state.The principal aim of our project was to induce the differentiation process of embryo-derived stem cells into neural cells (neurons, glial cells), to follow during the differentiation process the changing in the expression of characteristic stemness markers (OCT4, SOX2 and NANOG) responsible for the regulatory networks involved in embryo-derived stem cells pluripotency, whose understanding is fundamental for any potential therapeutic application. For this reason the use of advanced spectroscopic techniques, such as time-resolved fluorescence correlation spectroscopy (FCS), could allow to follow protein changes and to analyze different aspects such as the molecular dynamics and intracellular translocation of some selected transcription factors tightly bound to the activation of the ESCs differentiation processes into neural cells. Our Results show that the pluripotency circuit is known to act as a unit that strongly represses lineage specific gene expression in ESCs. However, rather than being a monolithic entity, the pluripotency circuit components have lineage specific roles, so that the same proteins can also be used for lineage selection.

The purpose of this study was to investigate the usefulness of the midcurricular HESI examination in identifying at-risk students early in their nursing program. The sample included baccalaureate nursing graduates from two university programs in the southeastern United States (n = 256). A quasiexperimental design was used to determine how well the midcurricular HESI predicted outcomes on the HESI E2 and the NCLEX-RN passing status while controlling for demographic and institutional covariates. The study used logistic regression and multiple linear regression to analyze the hypotheses. The midcurricular HESI examination was found to be a statistically significant predictor of NCLEX-RN outcomeboth before (P = .044) and after (P = .041) controlling for demographic factors. The study further found a statistically significant relationship between the midcurricular HESI and the HESI E 2 examinations (P < .001). In the post hoc analyses, students from the Accelerated and Fast Track degree programs scored significantly higher than did students in the Traditional Track on themidcurricular HESI examination. There were no statistically significant differences in HESI E2 scores or NCLEX-RN outcomes among the degree tracks. As anticipated, there was a statistically significant difference in both midcurricular HESI (P < .043) and HESI E2 (P < .016) scores between students who passed and those who failed NCLEX-RN. This study indicates that the midcurricular HESI examination is very useful in predicting outcomes in baccalaureate nursing education programs.

A LLE-GC-MS method was developed to detect PPCPs in surface water samples from Big Cypress National Park, Everglades National Park and Biscayne National Park in South Florida. The most frequently found PPCPs were caffeine, DEET and triclosan with detected maximum concentration of 169 ng/L, 27.9 ng/L and 10.9 ng/L, respectively. The detection frequencies of hormones were less than PPCPs. Detected maximal concentrations of estrone, 17β-estradiol, coprostan-3-ol, coprostane and coprostan-3-one were 5.98 ng/L, 3.34 ng/L, 16.5 ng/L, 13.5 ng/L and 6.79 ng/L, respectively. An ASE-SPE-GC-MS method was developed and applied to the analysis of the sediment and soil area where reclaimed water was used for irrigation. Most analytes were below detection limits, even though some of analytes were detected in the reclaimed water at relatively high concentrations corroborating the fact that PPCPs do not significantly partition to mineral phases. An online SPE-HPLC-APPI-MS/MS method and an online SPE-HPLC-HESI-MS/MS method were developed to analyze reclaimed water and drinking water samples. In the reclaimed water study, reclaimed water samples were collected from the sprinkler for a year-long period at Florida International University Biscayne Bay Campus, where reclaimed water was reused for irrigation. Analysis results showed that several analytes were continuously detected in all reclaimed water samples. Coprostanol, bisphenol A and DEET’s maximum concentration exceeded 10 µg/L (ppb). The four most frequently detected compounds were diphenhydramine (100%), DEET (98%), atenolol (98%) and carbamazepine (96%). In the study of drinking water, 54 tap water samples were collected from the Miami-Dade area. The maximum concentrations of salicylic acid, ibuprofen and DEET were 521 ng/L, 301 ng/L and 290 ng/L, respectively. The three most frequently detected compounds were DEET (93%), carbamazepine (43%) and salicylic acid (37%), respectively. Because the source of drinking water in Miami-Dade County is the relatively pristine Biscayne aquifer, these findings suggest the presence of wastewater intrusions into the delivery system or the onset of direct influence of surface waters into the shallow aquifer.

Those who are very wealthy may also be extremely free. Independently wealthy philanthropists epitomize this type of freedom. They seem to be able to act in whichever way they please, as long as they respect the limits of the law. Their freedom also implies that they do not experience as much accountability as other funders. Considering philanthropists’ ambitions as policymakers, and given their imposition of performance demands on their grantees, their accountability is relevant to investigate. However, there are no comprehensive comparative studies of philanthropists’ accountability, and there is mainly anecdotal evidence of a lack of accountability being derived from their independent wealth. This dissertation is a study of philanthropists’ accountability. I compare their experienced and exhibited accountability to that of other funders within societies, and I also compare philanthropists’ accountability across societies. I investigate whether philanthropists’ independent wealth influences to whom they are accountable, for what they are accountable, and how they are accountable. To learn about these topics, I examine their accountability relationships, their accountability mechanisms, and how they justify their potentially controversial funding of human embryonic stem cell research. Across these dimensions, I study their legal, financial, hierarchical, peer, professional, political, and fiduciary/social accountability. Empirically, I make a cross-sectional comparison of philanthropists to other funders of human embryonic stem cell research within and across three welfare regimes - liberal California, social democratic Sweden, and statist South Korea. I compare the accountability of independently wealthy philanthropists to that of public agencies, corporations, and fundraising dependent nonprofits. The empirical materials include 101 structured interviews with open-ended questions covering 51 funding organizations, as well as questionnaires explored in ANOVA and social network analysis. The study indicates that philanthropists experience and exhibit less accountability than other funders in some ways, in some contexts. By developing and using a framework to analyze their accountability, I show that philanthropists’ accountability is patterned within the societies in which they fund, and it differs greatly across societies. In California, philanthropists enact themselves as free actors, whereas in Sweden they enact a moral identity as funders of science. In South Korea, there is no clear boundary between philanthropic and corporate accountability. My results point to the contextual limits of philanthropists’ accountability. By enacting their moral identity in a way that conforms to local norms, philanthropists simultaneously retain and enable their continued freedom. In terms of their accountability, philanthropists are free to conform, and they become free by conforming.

Transwell inserts with microporous membranes, available from multiple commercial sources, have been widely used for various mammalian cell culture applications, including the reduction of cell culture mixing. In this study, we examined the feasibility and functionality of using this technology for separating human embryonic stem cells (hESCs) from their respective feeder cells. We found that when hESCs were propagated on transwell inserts positioned directly above feeder cells grown in a separate dish, the hESCs could be maintained in an undifferentiated state for over 10 passages with no change in their basic pluripotent characteristics. In parallel with our transwell insert experiments, we also evaluated the ability of a new defined, xeno-free medium, HEScGRO™, to enhance the animal-free characteristics of the transwell insert-based culture system. Results from our studies demonstrate that HEScGRO™ medium assists in maintaining the pluripotent characteristics of hESCs propagated in the transwell insert- based culture system. These combined results represent a significant development in properly segregating stem cells from their feeders, thus eliminating cell mixing, contamination, and providing the cells with a superior environment for nourishment and controlled self-renewal. Overall, this development in hESC propagation could have wide-reaching applications for self-renewal and differentiation studies within the field of stem cell biology.

Theses "The Tin Drum - the character of a child as a symbol of revolution, rebellion and war in the film" explores development of a child's character in film. Also in films about the war and revolution. (Go and see ,The White Ribbon, Alice in Wonderland, Threesomes of morals, Long live the Republic, etc ...) The work primarily analyzes the character of Oscar (The Tin Drum) and compares literary text and the movie. At the same time, the thesis further elaborates on the issue of film adaptations of literary works and also raises question of author's own projection into the main character. Although the focus is primarily on analysis of literary and cinematic form of The Tin Drum, this text also mentiones other key frames, on which different ways of working with child actors are portrayed.

The future of human embryonic stem cell (hESC) research with regards to their applicability in a therapeutic setting, relies on the development and standardisation of consistent and robust methods to demonstrate their defining characteristics; their pluripotent ability to form all three germ layers and their capacity for self-renewal. Although much research has been carried out to investigate new methods of culturing hESCs, many of these studies have not robustly concluded the impact of prolonged culture on genetic and genomic stability nor have they examined in any comparative detail the impact of the culture conditions such as differences in feeders used or the media composition in which the stem cells are cultured in. The aim of this thesis therefore was to investigate and evaluate methods for improving the uniform and robust culture and characterisation of hESCs over prolonged periods in culture. Four hESC lines ( RH5, HUES9, SHEF1 and NCL5) were chosen on the basis that they had not previously been well characterised and therefore could potentially benefit the wider stem cell community by increasing diversity, rather than continue to use the already small subset of well publicised lines. The RH5, HUES9, SHEF1 and NCL5 cells were subjected to long term passaging using recombinant enzyme TrypLE™ Express, on human feeders, mouse feeders and feeder free matrix Matrigel in combination with defined media mTeSR1, for uniform scale up. Changes in characteristic stem cell surface markers were compared using two techniques; flow cytometry and quantitative in situ fluorescence microscopy. Genomic stability was assessed by real time PCR. Chromosomal integrity was monitored using array genomic hybridisation (aCGH). Array genomic hybridisation analysis of cells cultured for 20 passages by enzymatic passaging revealed changes in copy number variations in all the stem cell lines. Aberrations on chromosomes 12, 17 and 20, appeared most commonly as a result of long term culture. Although no significant differences were seen between hESCs cultured on mouse and human feeders, cultures on Matrigel showed fewer detected chromosomal aberrations. Expression of cell surface stemness markers SSEA3, SSEA4, TRA1-60 and TRA1-81 were maintained by hESC cultured on all matrices and confirmed by the use of flow cytometry and high throughput quantitative immunofluorescence imaging using the TissueFaxs™ cell analysis microscopy system. In depth imaging revealed subtle but important differences in the way in which hESCs attach and proliferate on different matrices. Genetic profiling of each of the stem cell lines using Taqman Low density array cards to assess the expression of 96 genes by Real Time PCR, demonstrated the continued expression of stemness genes 21 at late passage, and low level expression of differentiation genes, inherent to particular stem cell lines. Although both mouse and human feeders and Matrigel support the undifferentiated growth of hESCs, subtle differences from the hESCs were seen as a result of their use, most obviously, changes in morphology and how they proliferate. This was further explored in the stem cell line NCL5, as it demonstrated a readiness to adapt to new matrices, better chromosomal stability and higher expression of cell surface markers compared with the other hESC lines. Using in vitro differentiation assays to all three germ layers, NCL5 cultured to late passage (p+20) on human feeder iMRC5, mouse feeder iMEF and feeder free matrix Matrigel, demonstrated the ability to differentiate to ectoderm, endoderm and mesoderm progenitors after induction using three 7 day flat based directed differentiation protocols. Altered differentiation patterns were detected by Real Time PCR and TissueFaxs™ imaging and quantitative analysis, as a consequence of the prolonged culture on the specific matrices used. Such key findings allude to the strong influences of microenvironment and will help to improve the standardisation of in vitro differentiation assays. From these studies, chromosomal changes had no impact on NCL5 stem cell lines‘ ability to form progenitors, however small genetic instabilities may still play a role in terminal differentiation of germ lineage specific cell types. The findings of the programme of work described has led to the successful culture methods and characterisation testing validated in this project being incorporated into routine culture and banking of research grade hESCs at the UK Stem Cell Bank. These protocols will now be made more widely available and should assist stem cell researchers in adopting the most suitable and optimum conditions for culturing stem cells in the undifferentiated and stable state. With the huge surge in stem cell research over the past decade, the development of robust characterisation and culture methods will undoubtedly have significant impact on the exploitation of these cells for regenerative medicine and to assist with this a future aim of the stem cell bank will be to standardise methodologies for clinical grade banking.

Development of safe and efficient approaches for gene delivery in human embryonic stem cells (hESc) and particularly in human induced pluripotent stem (hiPS) cells, which can be derived in a person-specific manner, is considered to be imperative for harnessing their full potential in both the basic and applied research. The aim of this study was to evaluate the potential of human artificial chromosome (HAC) for gene delivery and expression in hESc and hiPS cells. HAC offers many potential advantages including the provision for carrying large genes with corresponding regulatory elements to obtain long-term regulated gene expression. In addition, they can replicate and segregate independently without integration into the host cell genome. To develop HAC in hiPS cells, the first part of the study was aimed at generating hiPS cells utilising the Herpes Simplex Virus (HSV)-1 amplicon system. With the use of EBNA-1/OriP retention elements incorporated into the HSV-1 amplicon vectors, hiPS cells completely free of vector and transgenes sequences were successfully derived from human embryonic fibroblasts. The hiPS cells exhibited proliferation and differentiation potential similar to that of hESc. In the second part of the study, development of HAC in hESc and hiPS cells was assessed by utilising the HSV-1 amplicon system to deliver the HAC DNA. Analysis of the hESc confirmed the presence of functional HAC which replicated the behaviour of the host chromosomes. Additionally, HAC generation did not lead to impairment in the developmental potential and pluripotency of hESc. The hiPS cells supported HAC at low frequency but DNA also integrated into the host chromosomes. The HAC system, therefore, needs further refinements to improve the frequency of HAC formation and reduce the chromosomal integration of HAC constructs in hiPS cells. Overall, these findings provide a simple and safe way of pluripotency induction and genetic modification of pluripotent stem cells using the HSV-1 amplicon system and represent an important advance towards patient specific gene and cell therapy.

Prostate cancer is an important male health issue. The strategies used to diagnose and treat prostate cancer underscore the cell and molecular interactions that promote disease progression. Prostate cancer is histologically defined by increasingly undifferentiated tumour cells and therapeutically targeted by androgen ablation. Even as the normal glandular architecture of the adult prostate is lost, prostate cancer cells remain dependent on the androgen receptor (AR) for growth and survival. This project focused on androgen-regulated gene expression, altered cellular differentiation, and the nexus between these two concepts. The AR controls prostate development, homeostasis and cancer progression by regulating the expression of downstream genes. Kallikrein-related serine peptidases are prominent transcriptional targets of AR in the adult prostate. Kallikrein 3 (KLK3), which is commonly referred to as prostate-specific antigen, is the current serum biomarker for prostate cancer. Other kallikreins are potential adjunct biomarkers. As secreted proteases, kallikreins act through enzyme cascades that may modulate the prostate cancer microenvironment. Both as a panel of biomarkers and cascade of proteases, the roles of kallikreins are interconnected. Yet the expression and regulation of different kallikreins in prostate cancer has not been compared. In this study, a spectrum of prostate cell lines was used to evaluate the expression profile of all 15 members of the kallikrein family. A cluster of genes was co-ordinately expressed in androgenresponsive cell lines. This group of kallikreins included KLK2, 3, 4 and 15, which are located adjacent to one another at the centromeric end of the kallikrein locus. KLK14 was also of interest, because it was ubiquitously expressed among the prostate cell lines. Immunohistochemistry showed that these 5 kallikreins are co-expressed in benign and malignant prostate tissue. The androgen-regulated expression of KLK2 and KLK3 is well-characterised, but has not been compared with other kallikreins. Therefore, KLK2, 3, 4, 14 and 15 expression were all measured in time course and dose response experiments with androgens, AR-antagonist treatments, hormone deprivation experiments and cells transfected with AR siRNA. Collectively, these experiments demonstrated that prostatic kallikreins are specifically and directly regulated by the AR. The data also revealed that kallikrein genes are differentially regulated by androgens; KLK2 and KLK3 were strongly up-regulated, KLK4 and KLK15 were modestly up-regulated, and KLK14 was repressed. Notably, KLK14 is located at the telomeric end of the kallikrein locus, far away from the centromeric cluster of kallikreins that are stimulated by androgens. These results show that the expression of KLK2, 3, 4, 14 and 15 is maintained in prostate cancer, but that these genes exhibit different responses to androgens. This makes the kallikrein locus an ideal model to investigate AR signalling. The increasingly dedifferentiated phenotype of aggressive prostate cancer cells is accompanied by the re-expression of signalling molecules that are usually expressed during embryogenesis and foetal tissue development. The Wnt pathway is one developmental cascade that is reactivated in prostate cancer. The canonical Wnt cascade regulates the intracellular levels of β-catenin, a potent transcriptional co-activator of T-cell factor (TCF) transcription factors. Notably, β-catenin can also bind to the AR and synergistically stimulate androgen-mediated gene expression. This is at the expense of typical Wnt/TCF target genes, because the AR:β-catenin and TCF:β-catenin interactions are mutually exclusive. The effect of β-catenin on kallikrein expression was examined to further investigate the role of β-catenin in prostate cancer. Stable knockdown of β-catenin in LNCaP prostate cancer cells attenuated the androgen-regulated expression of KLK2, 3, 4 and 15, but not KLK14. To test whether KLK14 is instead a TCF:β-catenin target gene, the endogenous levels of β-catenin were increased by inhibiting its degradation. Although KLK14 expression was up-regulated by these treatments, siRNA knockdown of β-catenin demonstrated that this effect was independent of β-catenin. These results show that β-catenin is required for maximal expression of KLK2, 3, 4 and 15, but not KLK14. Developmental cells and tumour cells express a similar repertoire of signalling molecules, which means that these different cell types are responsive to one another. Previous reports have shown that stem cells and foetal tissues can reprogram aggressive cancer cells to less aggressive phenotypes by restoring the balance to developmental signalling pathways that are highly dysregulated in cancer. To investigate this phenomenon in prostate cancer, DU145 and PC-3 prostate cancer cells were cultured on matrices pre-conditioned with human embryonic stem cells (hESCs). Soft agar assays showed that prostate cancer cells exposed to hESC conditioned matrices had reduced clonogenicity compared with cells harvested from control matrices. A recent study demonstrated that this effect was partially due to hESC-derived Lefty, an antagonist of Nodal. A member of the transforming growth factor β (TGFβ) superfamily, Nodal regulates embryogenesis and is re-expressed in cancer. The role of Nodal in prostate cancer has not previously been reported. Therefore, the expression and function of the Nodal signalling pathway in prostate cancer was investigated. Western blots confirmed that Nodal is expressed in DU145 and PC-3 cells. Immunohistochemistry revealed greater expression of Nodal in malignant versus benign glands. Notably, the Nodal inhibitor, Lefty, was not expressed at the mRNA level in any prostate cell lines tested. The Nodal signalling pathway is functionally active in prostate cancer cells. Recombinant Nodal treatments triggered downstream phosphorylation of Smad2 in DU145 and LNCaP cells, and stably-transfected Nodal increased the clonogencity of LNCaP cells. Nodal was also found to modulate AR signalling. Nodal reduced the activity of an androgen-regulated KLK3 promoter construct in luciferase assays and attenuated the endogenous expression of AR target genes including prostatic kallikreins. These results demonstrate that Nodal is a novel example of a developmental signalling molecule that is reexpressed in prostate cancer and may have a functional role in prostate cancer progression. In summary, this project clarifies the role of androgens and changing cellular differentiation in prostate cancer by characterising the expression and function of the downstream genes encoding kallikrein-related serine proteases and Nodal. Furthermore, this study emphasises the similarities between prostate cancer and early development, and the crosstalk between developmental signalling pathways and the AR axis. The outcomes of this project also affirm the utility of the kallikrein locus as a model system to monitor tumour progression and the phenotype of prostate cancer cells.

This thesis analyses the dramatizations of larger epic units, which have appeared on the Czech national (or regional) shows of children's theatre since 1989. After characterizing the role of dramatic text created for the theatre played by children, the work attempts to establish the evaluation criteria for the dramatization of the investigated area. With regard to the artistic and educational objectives, the work compares the composition, language and thematic basis of selected texts. At the centre of attention are those means of adaptation which acquire particular significance especially in connection with a child actor; ways of creating adult characters, the depiction of the inner world of children's heroes etc. The main task of this text analysis is to answer the question about how far the authors were able to get within the narrow borders brought on by focusing on child actors up to fifteen years of age.

SOUSA, André Filipe Santos - Generation and characterization of functional sensory neurons from hESCs and hiPSCs. Coimbra : [s.n.], 2016. Dissertação de Mestrado em Biologia Celular e Molecular.
The lack of access to the neuronal tissue, limited our understanding about its development and physiology of pain in humans. Therefore, the generation of functional human sensory neurons for disease modelling, drug screening and clinical applications is an urgent and unmet need. Nociceptors are the sensory neurons related to sense the pain, characterized by the presence of transient receptor potential channels which have sensory functions linked to transduction of noxious stimuli as well as signalling within the pain system. In this thesis, we first characterized the nociceptors generated from induced pluripotent stem cell with a protocol developed on the laboratory, comparing it to a protocol described by Young et al., 2014. We used techniques as real-time reverse transcription-polymerase chain reaction and staining to check the neuronal phenotyping of the cells generated, as well as calcium imaging as a functional assay, to test about the functionality of transient receptor potential (TRP) channels, in order to characterize the neurons generated. We showed that only after 50 days of differentiation with both protocols we can have functional mature sensory neurons, with transient receptor potential cation channel sub family V, member 1 and transient receptor potential cation channel subfamily A, member 1 being activated. Moreover, the best results were achieved with the adapted protocol. Next, we focused on different approaches to improve the differentiation protocol. Changing cell line and replating dilution, did not enhance the protocol. Hence, we next hypothesized that current hurdles to speed up differentiation might be overcome by overexpression of key transcription factors involved in the sensory neurons differentiation: PR domain 12, brain-specific homeobox 3A, insulin gene enhancer and kruppel-link zinc finger transcription factor. A technique used on the laboratory, recombinase-mediated cassette exchange, was employed to generate two cell lines, one overexpressing PRDM12 and the other overexpressing ISL1-BRN3A-KLF7. We proved that in the new cell lines, the transcription factors were being correctly overexpressed, and collectively our results demonstrate that overexpressing PRDM12 improves the homogeneity of the cells generated. Further studies are required to evidence the impact of this new cell lines on the upgrading of the differentiation protocol. This work will open new opportunities for investigating in vitro disease modelling and evaluation of pharmacological responds to pain research.
A falta de acesso ao tecido neuronal limita o nosso conhecimento acerca do seu desenvolvimento e da fisiologia da dor nos humanos. Portanto, a geração de neurónios sensoriais humanos funcionais de forma à criação de modelos de doenças relacionadas com a dor, triagem de drogas e aplicações clinicas é uma necessidade urgente e não atendida. Os nociceptors são os neurónios sensoriais responsáveis por receber o estímulo da dor, caracterizados pela presença dos TRP channels que têm funções sensoriais na medida em que estão ligados à transdução de estímulos nocivos assim como à sinalização dentro do sistema da dor. Nesta tese, primeiramente caracterizamos os nociceptors gerados a partir de células estaminais recorrendo a um protocolo desenvolvido no nosso laboratório, comparando-o a um protocolo descrito por Young et al., 2014. Foram usadas técnicas como qRT-PCR e staining, de forma a conferirmos o fenótipo neuronal das células geradas, assim como calcium imaging como um teste funcional acerca da atividade dos TRP channels com o objetivo de caracterizarmos os neurónios gerados. Aqui, demonstramos que apenas após 50 dias de diferenciação com ambos protocolos conseguimos obter neurónios sensoriais maduros funcionais, com a ativação de TRPV1 e TRPA1. Ainda, o protocolo adaptado no laboratório teve melhores resultados. De seguida, focamo-nos nas várias formas de melhorarmos o protocolo de diferenciação. Mudar a linha celular usada e a diluição na fase de replating não aperfeiçoou o protocolo. Sendo assim, supomos que as barreiras em conseguir uma diferenciação mais rápida podiam ser ultrapassadas pelo aumento de expressão de fatores de transcrição envolvidas na diferenciação de neurónios sensoriais: PRDM12, BRN3A, ISL1 e KLF7. Uma técnica usada no laboratório, RMCE, foi usada de forma a gerar duas linhas celulares, uma com o aumento de expressão de PRDM12, e a outra com o aumento de expressão de ISL1, BRN3A e KLF7. Neste trabalho provamos que as novas linhas celulares estavam de facto com a expressão aumentada dos fatores de transcrição inseridos, e os nossos resultados demonstram que o aumento de expressão de PRDM12 contribuiu para o melhoramento da homogeneidade das células geradas. Futuros estudos têm de ser feitos de forma a evidenciar o impacto destas duas novas linhas celulares no aprimoramento do protocolo de diferenciação. Este trabalho vai abrir novas oportunidades para a investigação in vitro de modelos de doenças e a avaliação de respostas farmacológicas na investigação da dor.

Game theory is a branch of applied mathematics, whose main aim is to analyze various decision situations and predict their outcomes. This work applies game theory on three Jane Austen novels, namely Emma, Persuasion, and Pride and Prejudice. Analyzing the main topic in the plots (courtship, engagement and marriage) from the point of view of game theory and establishing the payoff matrices for each game described in the novel, we can observe several parallels in the three novels and the specifics of each of them. Respecting the limitations given by the nature of our source of information, this overall analysis allows us to reach conclusion about the predominant characteristics of the novels and proves applicability of game theory on literary work and theory.

European legal regulation of patents in the area of science and research Bioethics is an important part of law regulation in the medical field. According to the current state, bioethics is able to highlighted main issues, which are connected with medical research and suggest possible solution.This paper combines two controversial topics. First one is human embryonic stem cell research and second one is research on nanoparts and indicates Intelectual Property Law possibilities in this field. Paper is divided into two parts. First one deals with the legal regulation on research on embryo in the Czech Republic and in other states of The Western Europe. Main focus is based on patentability of research concerned with the human embryonic stem cells, which might have a great therapeutic potential but their preparation necessarily leads to the destruction of "human embryos". (HESC) Main concern is connected with regard to the European law and the current ground- breaking judgement, Brüstle v. Greenpeace eV. In mentioned judgment European Court of Justice held that after interpretation of the Directive on the legal protection of biotechnological inventions , it will not be able to grant a patent on research which led in the destruction of a human embryo. Paper also includes assessment of the attitude of the...

Praca składa się z wstępu, IV rozdziałów oraz uwag końcowych.W Rozdziale I nakreślony został kontekst naukowy tj. podstawowej jednostki życia jaką jest komórka (w tym możliwości praktycznego zastosowania komórek hESCs w leczeniu osób dotkniętych chorobami neurodegeneracyjnymi). Rozważania zawarte w tym rozdziale pozwolą Czytelnikowi zrozumieć procesy biologiczne, jakie zachodzą na najwcześniejszych etapach ludzkiego życia. Potrzeba uwzględnienia tej problematyki w niniejszej rozprawie wynika z obserwacji, że większość prac prawniczych ignoruje lub nie uwzględnia w wystarczającym stopniu aktualnego stanu wiedzy biomedycznej na temat początków ludzkiego życia. W Rozdziale II niniejszej rozprawy zaprezentowano krytyczną analizę statusu aksjologicznego ludzkiego zarodka oraz statusu ontologicznego komórek hESCs. Wśród mnogości postawionych w tej części pracy pytań, autor uznał za potrzebne udzielenie odpowiedzi na pytanie, jakim rodzajem bytu jest ludzki zarodek na płaszczyźnie teologicznej i filozoficznej? Czy przysługuje mu status osobowy czy też jest on innego rodzaju istotą żywą bądź stanowi tylko konglomerat komórek? Wobec faktu, że rozróżnienie to coraz częściej pojawia się w kodyfikacjach prawnych, a nie tylko w wybranych koncepcjach bioetycznych, zostało ono poddane gruntownej analizie. Obalając przy okazji analizy klonowania wiele mitów, przeprowadzono rozważania etyczno-medyczne dotyczące najnowszych zdobyczy biotechnologii, m.in. dotyczących celowego modyfikowania genów. W Rozdziale III, omówiono ogólne kwestie bioetyczne, wiążące się z tytułowym zagadnieniem, w tym poddano analizie pojęcie godności w wybranych porządkach prawnych i wpływu rozumienia tego pojęcia na prawo do życia. Przeanalizowano obowiązujące w tym zakresie standardy, m.in. obowiązujące w ramach regionalnego systemu ochrony praw człowieka (a zwłaszcza wynikające z Konwencji o Ochronie Praw Człowieka i Godności Istoty Ludzkiej wobec zastosowań Biologii i Medycyny). Wprawdzie tzw. Konwencja Bioetyczna nie obowiązuje ani w Polsce ani w żadnym z będących przedmiotem analizy prawnoporównawczej porządków prawnych, to jej przepisy są na tyle istotne, że nie sposób ich pominąć w rozważaniach. Uwzględniono także dokumenty Unii Europejskiej, jak i wybrane orzeczenia TSUE. Dodatkowo dokonano także przeglądu regulacji prawnych związanych ze szczególnym rodzajem produktów leczniczych opartych na komórkach hESCs (ang. Advanced Teraphy Medicinal Products, ATMP). Kluczowe zagadnienia zostały omówione w Rozdziale IV, będącym najbardziej obszerną częścią pracy. Znalazła się w nim bowiem zarówno analiza obowiązującego w Polsce stanu prawnego, jak też prawnoporównawcza przyjętych w wybranych krajach rozwiązań w dziedzinie prokreacji pozaustrojowej: Podjęto tam też próbę udzielenia odpowiedzi na dwa następujące pytania: Po pierwsze, jak daleko powinny sięgać granice kryminalizacji w analizowanej dziedzinie oraz po drugie, jak przedstawiają się one w świetle obowiązującego prawa? Aby lepiej zrozumieć kształtowanie się społecznej świadomości co do medycznego wykorzystania komórek hESCs, dokonano krótkiego rysu historycznego prawa do aborcji na przykładzie wybranych porządków krajowych. Następnie zestawiono z sobą dwa systemy prawa uznawane pod wieloma względami za najbardziej liberalne i najbardziej rygorystyczne w Europie: odpowiednio – brytyjski i niemiecki. Wobec faktu, że praca obejmuje kilka systemów prawa, część porównawcza nie aspiruje do wyczerpania problematyki, jaką nasuwa interpretacja przedstawionych przepisów, a jedynie zmierza do wskazania kierunków kryminalizacji i objętych nią obszarów. Z podobnego powodu na gruncie prawa polskiego niektóre kwestie zostały tylko zasygnalizowane. Poddano natomiast szerszej analizie przepisy karne ustawy o leczeniu niepłodności, które dotyczą ludzkiego zarodka oraz procedury in vitro. W związku z tym, że terapia z użyciem komórek hESCs wciąż uznawana jest za eksperyment medyczny, w ramach postulatów de lege lata zaproponowano wyodrębnienie w polskim prawie terapii innowacyjnej oraz terapii eksperymentalnej, zakładając, że wbrew pozorom nie stanowią one synonimów, krytycznie odnosząc się do obowiązujących regulacji prawnych w tym zakresie.
Research with human embryonic stem cells (hESCs) is highly debated, evoking strong opinions from all sectors of society. Both sides of the debate are interested in protecting human life, so why are their views so conflicted? It comes down to how the human blastocyst is viewed. Some people see destroying a blastocyst for its cells as destroying an unborn child. Others feel that a blastocyst does not constitute a human life, because unless a blastocyst embeds in the uterus wall, it will never have the chance to develop into a baby. So which moral principle has the upper hand in this situation? The answer hinges on how we view the embryo. Does it have the status of a person? The main viewpoints are outlined below: 1. the embryo has full moral status from fertilisation on wards; 2. Human status is not attained until 14 days post fertilisation; 3. the embryo has increasing status as it develops; 4. the embryo has no moral status at all. Different religions view the status of the early human embryo in different ways. For example, the Roman Catholic church believes the embryo has the status of a human from conception and no embryo research should be permitted. Other religions take a different stance. Judaism and Islam emphasise the importance of helping others and argue that the embryo does not have full human status before 40 days, so both these religions permit some research on embryos. It forces us to choose between two moral dilemmas: the duty to prevent or alleviate suffering (the early embryo has to be destroyed but could potentially aid the discovery of new medical treatments that would alleviate the suffering of many people) or the duty to respect the value of human life. A natural question is where does the middle ground lie? This is where discussion is vitally important. Debates and discussions about the moral and ethical status of hESCs help establish the rules and regulations that govern scientific research and the development of medical treatments utilising stem cells. In reference to hESCs cells, the main intention was to analyze legal mechanisms that were created in Poland to regulate this issue and attempt to assess their effectiveness. While simultaneously reviewing international standards as a means to compare and contrast. The main issues surrounding the use of hESCs cells was supplemented with a discussion of national regulations (and their sources) regarding the protection of the human life and the right to abortion. Standards were analysed, including the regional system of human rights protection (in particular those arising from the Convention for the Protection of Human Rights and Human Dignity in relation to the applications of Biology and Medicine). Admittedly, The Bioethical Convention does not apply in Poland or in any of the legal systems that have formed the basis of the comparative legal analysis. However its provisions are so important that they cannot be omitted (see: art. 18 Convention). European Union documents as well as selected judgements of the CJEU were also taken into account. Additionally, legal regulations related to special types of medicinal products based on hESCs - Advanced Therapy Medicinal Products (ATMP) were reviewed. Key issues are discussed in the last chapter, which is the most extensive part of the thesis. It contains existing analysis and legal status in Poland, as well as comparing laws adopted in selected countries (Britain and Germany respectively) pertaining to IVF. Due to the fact that therapy with hESCs cells is still considered a medical experiment, de lege lata postulates separating innovative therapy and experimental therapy in Polish law, assuming that, contrary to appearances, they are not synonymous. The comparative analysis revealed that while the legal status of the human embryo is different in each country. There is a clear tendency to treat it, at least in its initial stage of development as a carrier of genetic information rather than a separate human entity. However, there is a general consensus that the human embryo has a special place in the field of law. Giving it greater value than tissue fragments or other biological materials of human origin, but smaller than that attributed to a fully developed human being. Therefore, it is reasonable to say that the law does not have to qualify the legal status of an embryo, but should clearly define what can and cannot be done with it at particular stages of its development. In the dissertation, it was found that the national law of the countries studied, and especially international law, regulates the title issue in a minimal and often indirect way. A similar situation occurs in Poland, as there are no regulations that would comprehensively regulate the manner of dealing with hESCs. Some information on this subject is provided to us by the Act on Infertility Treatment. The provisions of this law criminalize (in the context of IVF procedure) the destruction of an embryo capable of proper development (art. 83). They can be used for medical tests/research requiring hESCS. It can be assumed that unless the hESCs are taken from the germ node, it will not be destroyed, this act will not be criminalized. For example, you can point to the so-called Transplant Act, the provisions of which explicitly exclude hESCs from its scope (art. 1.2). The consequence of such an exclusion is among others, the fact that there is no special body in Poland that would directly supervise research using this type of biological material. The possible admissibility of research on embryonic stem cells in Poland can also be attributed to the Penal Code relating to the protection of the beginnings of human life and regulations regarding scientific experiments. Comparative legal research has shown that a completely different legal picture prevails in Germany. Pursuant to the ESchG Act, any research on embryos obtained by the in vitro method and originating from national health centers was prohibited, as opposed to tests performed on embryos transported from foreign centers. The regulations of this law are characterized by a certain hypocrisy of German legislation (since the ban applies only to embryos established in Germany), but at the same time testify to its pragmatism. This prohibition does not apply to embryos in the pronucleus stage. Different regulations apply in the United Kingdom, where according to HFEAct 1990 and 2008 and the Regulation on Human Fertilization and Embryology of 2001, both the use of surplus embryos and their creation specifically for research purposes is allowed, but not later than 14 days on their development.