This master’s thesis is aimed at practical use of fundamental analysis of stocks. Its main goal is, on the basis of defined procedures, to analyze and valorize companies ČEZ, a. s., ECM Real Estate Investments A. G. and then decide about possible investments into the stocks of these companies. This thesis is divided into two main parts. First part deals with theoretical solutions of fundamental analysis and brief comment of another two ways how to analyze stocks – graphic and psychological analysis. In second part there is a practical elaboration of fundamental analysis for aforesaid companies and, in the end of this thesis, there is a summary of achieved results with brief commentary for possible investors.

Deciphering exogenous cues that determine stem cell fate decisions is a persisting challenge of cell biology and bioengineering. In an effort to unravel the role of spatial constraints in the cell-instructive characteristics of bone marrow microenvironments, murine hematopoietic stem and progenitor cells (HSPC) were exposed to fibronectin-coated microcavities in vitro. Microcavity sizes were chosen to allow for the inclusion of either individual or multiple cells. Repopulation experiments using lethally irradiated mice showed that the maintenance of functional HSPC in culture critically depends on cavity dimensions. Short-term repopulating hematopoietic stem cells (ST-HSC) were found to be best supported within single-cell sized compartments while long-term repopulating HSC (LT-HSC) were maintained within both cavity sizes. In sum, the reported data reveal spatial restriction to be a simple but powerful means for directing HSPC fate ex vivo.

The present study focuses on the cardiac function of Drosophila melanogaster. Drosophila heart parameters are evolutionarily conserved, making Drosophila a useful human heart disease model. This model enables the in vivo investigation of physiological and genetic methods. This thesis is subdivided into four parts: parts 1-3 comprise the introductions of three publications, and part 4 presents unpublished data. The first publication is about the heart physiology of Drosophila. It explains how intracardiac valve cells work and proves their participation in blood flow directionality. A databased model shows the orientation of myofibrils within the valve cell. The myofibrils allow the valve cells to oscillate between a roundish and elongated cell shape. A toll-GFP enhancer line was shown to mediate strong reporter gene activity in the intracardiac valve of third instar larvae, pupae and adults. Transmission electron microscopy (TEM) analyses and immunohistochemical studies showed the differentiation of larval valve cells for the first time. The second publication focuses on the cardiac extracellular matrix (ECM), which contains two unique proteins - Lonely heart (Loh) and Pericardin (Prc). The study demonstrated that Loh is crucial for Prc recruitment to the developing matrix. Loh is anchored to the ECM by its thrombospondin type 1 repeat (TSR1-1) with its embedded putative glycosaminoglycan (GAG)-binding side. The N-terminus of Loh is proposed to face the plasma membrane. Prc is presumably recruited by two Loh TSR1 domains (TSR1-2 and TSR1-4). Nearly all Drosophila tissues, except salivary glands, create Prc networks through ectopic Loh expression. The study also found that the amount of Prc and Loh in the cardiac ECM influences heart function. The third publication investigated a set of neuropeptides and their ability to modulate cardiac function in third instar larvae. The results showed that 11 of the 19 tested peptides significantly affected the heart function in semi-intact larvae. Furthermore, the peptides’ in vivo relevance was tested through the knockdown of chronotropic peptide precursors. The study found that a RNAi mediated knockdown of all respective peptide precursors affected the heart rate. By combining semi-intact heart preparations and in vivo analyses, we identified several heartbeat-modulatory peptides in Drosophila. The unpublished data introduces a new software program called HIRO. It is written in Java, platform-independent and can easily detect the heart rhythm. Only mild anaesthesia and basic equipment are needed to record the Drosophila heartbeat. HIRO was used to show the influence of the RNAi-mediated downregulation of critical ECM proteins in Drosophila third instar larvae. The screen revealed Myospheroid and Laminin A as promising candidates that can significantly affect the heart parameters. HIRO is optimised for future applications and can be used as a high-throughput screening software with a simple setup. Taken together, this thesis provides new insights into the physiology and function of the Drosophila heart. The developed software HIRO comes with a user-friendly interface and a step-by-step introduction to easily conduct heart parameter measurements. HIRO will help to expand our knowledge of the fundamental processes in the model organism Drosophila melanogaster.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / Every year, small- and medium-sized enterprises (SMEs) expand their participation in the Peruvian market, while facing high internal disorganization issues that stifle their growth. This problem is rooted on the fact that the contents generated by these companies are not usually adequately recorded, managed, and exploited, and thus negatively affecting the organization and their competitiveness levels. Now, although most of this issue may be solved through enterprise content management (ECM) solutions, they are not affordable for most SMEs because of their high prices. Within this context, this study proposes the implementation of a cloud-based open-source ECM model at a manufacturing SME in Peru. Through this model, the company was able to access the benefits of an ECM to restructure the way they manage content, gaining 67% more efficiency, establishing a collaboration channel between employees, suppliers, and customers, and reporting a 93% model adaptation rate among staff members. / Revisión por pares

Remodeling after myocardial infarction (MI) associates with left ventricular (LV) dilation, decreased cardiac function and increased mortality. The dynamic synthesis and breakdown of extracellular matrix (ECM) proteins play a significant role in myocardial remodeling post-MI. Expression of osteopontin (OPN) increases in the heart post-MI. Evidence has been provided that lack of OPN induces LV dilation which associates with decreased collagen synthesis and deposition. Inhibition of matrix metalloproteinases, key players in ECM remodeling process post-MI, increased ECM deposition (fibrosis) and improved LV function in mice lacking OPN after MI. This review summarizes — 1) signaling pathways leading to increased expression of OPN in the heart; 2) the alterations in the structure and function of the heart post-MI in mice lacking OPN; and 3) mechanisms involved in OPN-mediated ECM remodeling post-MI.

Background: STAT3 is increasingly becoming known for its non-transcriptional regulation of mitochondrial bioenergetic function upon activation of its S727 residue (S727-STAT3). Lengthy mitochondrial dysfunction can lead to cell death. We tested whether an integrin-FAK-STAT3 signaling pathway we recently discovered regulates mitochondrial function and cell survival, and treatments thereof. Methods: Cultured mouse brain bEnd5 endothelial cells were treated with integrin, FAK or STAT3 inhibitors, FAK siRNA, as well as integrin and STAT3 activators. STAT3 null cells were transfected with mutant STAT3 plasmids. Outcome measures included oxygen consumption rate for mitochondrial bioenergetics, Western blotting for protein phosphorylation, mitochondrial membrane potential for mitochondrial integrity, ROS production, and cell counts. Results: Vitronectin-dependent mitochondrial basal respiration, ATP production, and maximum reserve and respiratory capacities were suppressed within 4 h by RGD and αvβ3 integrin antagonist peptides. Conversely, integrin ligands vitronectin, laminin and fibronectin stimulated mitochondrial function. Pharmacological inhibition of FAK completely abolished mitochondrial function within 4 h while FAK siRNA treatments confirmed the specificity of FAK signaling. WT, but not S727A functionally dead mutant STAT3, rescued bioenergetics in cells made null for STAT3 using CRISPR-Cas9. STAT3 inhibition with stattic in whole cells rapidly reduced mitochondrial function and mitochondrial pS727-STAT3. Stattic treatment of isolated mitochondria did not reduce pS727 whereas more was detected upon phosphatase inhibition. This suggests that S727-STAT3 is activated in the cytoplasm and is short-lived upon translocation to the mitochondria. FAK inhibition reduced pS727-STAT3 within mitochondria and reduced mitochondrial function in a non-transcriptional manner, as shown by co-treatment with actinomycin. Treatment with the small molecule bryostatin-1 or hepatocyte growth factor (HGF), which indirectly activate S727-STAT3, preserved mitochondrial function during FAK inhibition, but failed in the presence of the STAT3 inhibitor. FAK inhibition induced loss of mitochondrial membrane potential, which was counteracted by bryostatin, and increased superoxide and hydrogen peroxide production. Bryostatin and HGF reduced the substantial cell death caused by FAK inhibition over a 24 h period. Conclusion: These data suggest that extracellular matrix molecules promote STAT3-dependent mitochondrial function and cell survival through integrin-FAK signaling. We furthermore show a new treatment strategy for cell survival using S727-STAT3 activators.

Oxidation of polyunsaturated fatty acids contributes to different aspects of the inflammatory response due to the variety of products generated. Specifically, the oxidation of DHA produces the end-product, carboxyethylpyrrole (CEP), which forms a covalent adduct with proteins via an ϵ-amino group of lysines. Previously, we found that CEP formation is dramatically increased in inflamed tissue and CEP-modified albumin and fibrinogen became ligands for αDß2 (CD11d/CD18) and αMß2 (CD11b/CD18) integrins. In this study, we evaluated the effect of extracellular matrix (ECM) modification with CEP on the adhesive properties of M1-polarized macrophages, particularly during chronic inflammation. Using digested atherosclerotic lesions and in vitro oxidation assays, we demonstrated the ability of ECM proteins to form adducts with CEP, particularly, DHA oxidation leads to the formation of CEP adducts with collagen IV and laminin, but not with collagen I. Using integrin αDß2-transfected HEK293 cells, WT, and αD-/- mouse M1- polarized macrophages, we revealed that CEP-modified proteins support stronger cell adhesion and spreading when compared with natural ECM ligands such as collagen IV, laminin, and fibrinogen. Integrin αDß2 is critical for M1 macrophage adhesion to CEP. Based on biolayer interferometry results, the isolated αD I-domain demonstrates markedly higher binding affinity to CEP compared to the “natural” αDß2 ligand fibrinogen. Finally, the presence of CEP-modified proteins in a 3D fibrin matrix significantly increased M1 macrophage retention. Therefore, CEP modification converts ECM proteins to αDß2- recognition ligands by changing a positively charged lysine to negatively charged CEP, which increases M1 macrophage adhesion to ECM and promotes macrophage retention during detrimental inflammation, autoimmunity, and chronic inflammation.

Damage to our tissues occurs daily and must be repaired by the body in a timely manner in order to prevent infection and restore tissue integrity. Many cell types are involved in the healing process, but it is the cells of the stroma that are largely responsible for rebuilding fibrous tissue, which provides structure and support for all other cell types during healing. This dissertation focuses on stromal tissue repair, the rebuilding of fibrous tissue by fibroblasts following injury. Specifically, I focus on 1) models to study wound healing in vitro, and the specific biological processes of healing that each model captures, 2) the response of engineered stromal microtissues to different methods of injury, namely laceration and laser ablation, and the subsequent clearance and rebuilding of the extracellular matrix by fibroblasts, and 3) how different types of stromal cells and extracellular matrix proteins contribute to tissue repair in vitro.

The cardiovascular system of Drosophila melanogaster consists of a cardiac tube composed of myogenic cardiomyocytes and associating non-contractile pericardial cells, pumping hemolymph into the open circulatory system. The cardiac tube, known as the dorsal vessel, is embedded in a highly regulated extracellular matrix environment, required to maintain cellular integrity and cardiac function. After embryogenesis, the dorsal vessel undergoes extensive physiological changes, relying on the extracellular matrix to adapt and remodel accordingly. Three extracellular matrix proteins are investigated throughout this thesis: Type IV Collagen, Laminin and Pericardin. Due to their localization, morphology, and role in early development, the pericardial cells are candidate cells responsible for dorsal vessel extracellular matrix deposition and regulation throughout post-embryonic growth. Using immunofluorescence techniques in combination with confocal microscopy, I characterize the association between pericardial cells and extracellular matrix proteins, and quantify extracellular matrix protein deposition at the dorsal vessel throughout post-embryonic development. Gene knock-down experiments assess pericardial cell contribution to extracellular matrix synthesis and deposition at the dorsal vessel in third instar larva. Moreover, I quantify extracellular matrix protein deposition at the dorsal vessel in the absence of pericardial cells. These data suggests that pericardial cells regulate extracellular matrix protein deposition, localization and contribute to proper cardiac morphology in post-embryonic development. / Thesis / Master of Science (MSc)

This dissertation concerns the alteration of functional connectivity in the human brain through different patterns of somatosensory stimulation. In particular, I distinguish whether stimuli are regular (i.e., expected by the subject) or irregular (i.e., unexpected by the subject). An emerging theory of brain function known as Predictive Coding states that the brain is continuously creating an internal model of its environment that is constantly trying to predict what is going to happen. Expected sensory input leads to model consol- idation, while unexpected input leads to model update. In this context it is assumed that central neuronal processing differs significantly between these two cases. Furthermore, in my experiments, the stimulation is applied in two more variants which are also believed to be processed in completely different ways: consciously perceptible (suprathreshold) and imperceptible (subthreshold). To measure functional connectivity in the acquired fMRI data, a method referred to as eigenvector centrality mapping (ECM) was chosen. This method has gained increasing attention in the fMRI community, as it represents a whole-brain approach that can be ap- plied for resting-state experiments. While there are a number of other centrality measures, each with their advantages and disadvantages, ECM stands out as being parameter-free and does not depend on prior assumptions. Similar to Google’s Pagerank algorithm, it assigns areas (“nodes”) in a network with a high centrality score that are closely connected to other central areas as well. Generally, increased connectivity is interpreted as of greater “importance” to the network. As there are different approaches on how to calculate ECM, I critically examine these and delve deeper into the method itself. Three main research questions guided this study: 1. Is there a brain connectivity (ECM) alteration in the human brain for somatosensory stimulation that is pattern dependent (7 Hz irregular vs. regular)? 2. Is there a brain connectivity (ECM) alteration in the human brain for somatosensory stimulation that is intensity dependent (7 Hz suprathreshold vs. subthreshold)? 3. Are these different somatosensory stimulations (subthreshold, suprathreshold, irregular, regular) accompanied by a subsequent behavioral change? Two experiments were conducted. In Experiment 1, participants were exposed to all four stimulation conditions consecutively. Results showed significant ECM alterations compared to the initial baseline, suggesting persisting effects. To counter this, Experiment 2 adopted a different approach. Here, individual stimulations were applied to separate groups, with an additional control group for comparison. The results from Experiment 2 revealed that irregular stimulation compared to regular showed decreased connectivity in specific brain regions, aligning with the Predictive Coding theory. Suprathreshold stimulation showed increased connectivity in areas related to sensory input integration, possibly linked to conscious perception. Furthermore, all participants, regardless of stimulation type, showed heightened connectivity in somatosensory regions, suggesting a shared focus on tactile anticipation. The behavioral session from Experiment 2 found that irregular suprathreshold stimulation led to a decreased sensitivity to near-threshold stimuli. However, this change wasn't mirrored in the functional connectivity data. In conclusion, this research validated the differential neural processing of various somatosensory stimulations, supporting the Predictive Coding theory. The study also underscored the challenges and considerations in using ECM, particularly urging caution with methods that combine positive and negative correlations.:1 - Personal Motivation 2 - Introduction 3 - Background 3.1 Cognitive Neuroscience 3.2 Predictive Coding and the Free Energy Principle 3.3 Functional Magnetic Resonance Imaging (fMRI) 3.4 Blood-Oxygen Level Dependent (BOLD) Signal 3.5 Resting-State Functional Connectivity (RSFC) 3.6 Eigenvector Centrality Mapping (ECM) 3.7 Previous ECM Experiments - an Overview 3.8 Statistical Remarks 4 - Materials and Methods 4.1 Experimental Setup 4.1.1 Subjects 4.1.2 Experimental Procedures 4.1.2.1 Electrical stimulation 4.1.2.2 Absolute detection threshold examination 4.1.2.3 fMRI and behavioral data acquisition 4.2 fMRI Data Preprocessing 4.2.1 Prior Steps 4.2.2 Slice Time Correction 4.2.3 Motion Correction 4.2.4 Coregistration, Segmentation and Normalization 4.2.5 Spatial Filtering 4.2.6 Temporal Filtering 4.2.7 Grey Matter, White Matter and Cerebrospinal Fluid Masking 4.2.8 Nuisance Regression 4.3 ECM Approaches 4.4 Flexible Factorial Design 4.5 Seed-Based Functional Connectivity Analysis 5 - Results 5.1 Experiment 1 5.1.1 Detailed Results 5.1.2 Summary Experiment 1 5.2 Experiment 2 - fMRI Session 5.2.1 Detailed Results - ADD Approach 5.2.2 Detailed REsults - POS / NEG / ABS approach 5.2.3 Summary Experiment 2 5.3 ECM Approach Differences Illustrated by Examples 5.4 Seed-Based Functional Connectivity Analysis 5.5 Distribution of Voxel Timie Series Correlation Foefficients 5.6 Experiment 2 - Behavioral Session 6 - Discussion 6.1 Interpretation of Experimental Results 6.1.1 Experiment 1 6.1.2 Experimemnt 2 - fMRI Session 6.1.3 Experiment 2 - Behavioral Session 6.2 ECM Approaches 6.3 Further Considerations and Future Outlook 7 - Conclusion