At sites of vascular injury, the critical blood clotting enzyme thrombin is generated from prothrombin via Prothrombinase, a macromolecular, Ca2+-dependent enzymatic complex consisting of the serine protease factor Xa and the non-enzymatic cofactor factor Va, assembled on the membranes of activated platelets. Platelets regulate thrombin formation by providing specific binding sites for the components of Prothrombinase and by releasing a platelet-derived factor V/Va molecule that is more procoagulant than its plasma counterpart and partially resistant to proteolytic inactivation. This dissertation identifies and characterizes the subpopulation of platelet-derived factor V/Va that is responsible for the observed protease resistance, and the mechanism by which Prothrombinase bound to platelets differs from a model system using vesicles composed of 75% phosphatidylcholine (PC) and 25% phosphatidylserine (PS), PCPS vesicles. Previous studies have demonstrated that activated platelets release a dissociable pool of factor V/Va and a non-dissociable, membrane-bound pool, which is covalently attached to the platelet membrane through a glycosylphosphatidyl inositol (GPI) anchor. Data described herein demonstrate unequivocally that the pool of platelet-derived factor V/Va that is resistant to proteolytic inactivation by activated protein C is provided exclusively by the non-dissociable GPI-anchored pool. Further, although this factor Va pool is susceptible to proteolysis by plasmin, the fragments formed are associated with sustained and increased cofactor activity. These observations indicate that tethering of factor Va to the membrane surface via a GPI anchor imparts resistance to proteolytic inactivation and sustained thrombin generation at sites of vascular injury. For several years it has been known that Prothrombinase assembled on PCPS vesicles does not mimic that bound to platelets. While both enzymes cleave prothrombin at Arg271 and Arg320 to form thrombin, prothrombin activation proceeds via the prethrombin-2 pathway (initial cleavage at Arg271) on the platelet surface, in contrast to the meizothrombin pathway (initial cleavage at Arg320) on PCPS vesicles. Using thrombin active site inhibitors, we demonstrate that the preference for either pathway is dictated by the conformation in which prothrombin is bound by the membrane-bound enzyme. The prethrombin-2 pathway of prothrombin activation catalyzed by platelet-bound Prothrombinase is a direct consequence of configuring prothrombin in a proteinase-like state resulting in the exposure of a pseudo-active site that can be stabilized by active site thrombin inhibitors. Conversely, prothrombin is preferentially configured in the zymogen-like state on PCPS vesicles where the meizothrombin pathway is preferred. Additional support for the differential assembly of Prothrombinase on the platelet surface is provided by observations made using prethrombin-1, an intermediate formed by cleavage of prothrombin at Arg155 by the formed thrombin. Prethrombin-1 is converted into fragment-2 and thrombin by platelet-bound Prothrombinase at a substantially higher rate than vesicle-bound Prothrombinase. The decreased rate of prethrombin-1 activation in the model system is due, in part, to inhibition of the vesicle-bound enzyme by the fragment-2 generated in the reaction. Taken together, these data not only provide important molecular insights into the mechanisms by which Prothrombinase bound to activated platelets at sites of vascular injury regulates the procoagulant response to effectively support robust thrombin generation, but also provides potential mechanistic sites that could be targeted therapeutically.

In this project, the correction factor of different solar panels of the laboratory of the University of Gävle, located in Sweden, is going to evaluated. The solar modules’working conditions are different from the ones used to test them in the laboratory. In the laboratory. the output energy of the modules is less than in working conditions,and therefore a correction factor is going to be calculated from the data collected, inorder to describe the factors that affect the performance of the solar modules.Also, the obtained correction factor validity for different PV systems it is going to be examined, determining which system has a better correction factor and the energy losses due to temperature, angle of incidence and micro invertor.

OBJETIVO: Identificar las principales complicaciones fetales y del recién nacido así como los factores de riesgo de la Isoinmunización Rh. DISEÑO: Estudio Descriptivo Analítico Retrospectivo. LUGAR: Instituto Especializado Materno Perinatal, MINSA. MATERIAL Y MÉTODO: 116 gestantes Rh negativo que se atendieron el 0000maternas y neonatales para determinar las diferencias entre los casos de grupo control se utilizaron las pruebas de significación estadísticas Chi2 y prueba t de Studen. RESULTADOS: De 54,418 partos atendidos en la institución durante este período, se obtuvieron 116 (0,21%) madres Rh negativo que cumplieron con los criterios de inclusión, de las cuales el 12,1%(14) presentaron isoinmunización Rh. La edad promedio de las madres sensibilizadas fue de 30 años, se observo una mayor incidencia en multíparas (50%) y un menor número de control pre natal (1-3) en comparación de las madres sensibilizadas (>4). El 50% de las madres sensibilizadas presentaron antecedentes de aborto, 14,3% antecedentes de óbito y de transfusión y el 21,4% antecedente de hidrops y kernicterus; Siendo estas variables factores de riesgo de presentar isoinmunización. La tasa de cesárea fue de 78,6%, se encontró mayor porcentaje de partos prematuros en la madres sensibilizadas. El Apgar promedio en los recién nacidos fue de 6 al minuto y 8 a los 5minutos. Las complicaciones fetales más frecuentes fueron: ictericia anemia e hidrops y la mortalidad fetal fue de 7,1%(1). CONCLUSIONES: Es necesario evaluar la prevención mediante los estudios de los factores de riesgo teniendo en consideración que los niños que presentaron isoinmunización Rh se encontraron en el grupo en el cual la madre no recibió gammaglobulina anti-D profiláctica y que por lo tanto no tuvieron un adecuado control prenatal en anteriores embarazos ya sea por el factor económico, por el factor ignorancia o por la poca cobertura de éste. Por ello es necesario establecer políticas dirigidas a la promoción y prevención. Palabras claves: isoinmunización Rh, factores de riesgo, complicación fetal y neonatal.

Previous systematic reviews indicate a lack of reporting of reliability and validity evidence in subsets of the medical education literature. Psychology and general education reviews of factor analysis also indicate gaps between current and best practices; yet, a comprehensive review of exploratory factor analysis in instrument development across the continuum of medical education had not been previously identified. Therefore, the purpose for this study was critical review of instrument development articles employing exploratory factor or principal component analysis published in medical education (2006-2010) to describe and assess the reporting of methods and validity evidence based on the Standards for Educational and Psychological Testing and factor analysis best practices. Data extraction of 64 articles measuring a variety of constructs that have been published throughout the peer-reviewed medical education literature indicate significant errors in the translation of exploratory factor analysis best practices to current practice. Further, techniques for establishing validity evidence tend to derive from a limited scope of methods including reliability statistics to support internal structure and support for test content. Instruments reviewed for this study lacked supporting evidence based on relationships with other variables and response process, and evidence based on consequences of testing was not evident. Findings suggest a need for further professional development within the medical education researcher community related to 1) appropriate factor analysis methodology and reporting and 2) the importance of pursuing multiple sources of reliability and validity evidence to construct a well-supported argument for the inferences made from the instrument. Medical education researchers and educators should be cautious in adopting instruments from the literature and carefully review available evidence. Finally, editors and reviewers are encouraged to recognize this gap in best practices and subsequently to promote instrument development research that is more consistent through the peer-review process.

Under certain loading conditions, surfaces topography coupled with materials degree of brittleness can significantly compromise the mechanical performance of structures. The foregoing remains valid even if roughness is intentionally introduced for engineering reasons. In either case, stress can concentrate. The case of the stress concentration in surfaces having randomly distributed pits is a problem that, although being very practical, yet it remains unsolved. The complexity of a random configuration renders difficult the problem of analytically finding relationships between surface parameters and markers indicative of mechanical failure. Another difficulty is the reproducibility of replicates of specimens possessing random rough surfaces, for destructive testing followed by statistical analysis. An experimental technique to produce highly controlled replicates of random rough surfaces (including modeling of degradation growth) was developed. This method was used to experimentally and statistically study the effects on fracture of early randomly degraded surfaces of poly methyl methacrylate (PMMA) versus topographical parameters. Growth of degradation was assumed to go from an engineering surface to one whose heights are normally distributed. (Early stage of degradation is meant to be that level of roughness which is in the neighborhood of the critical flaw size for a given material). Among other findings, it was found that neither stress nor strain alone can be used to predict fracture at this early stage of degradation. However, fracture location was found to be strongly correlated to the ratio of the root-mean square roughness (RMS) to auto correlation length (ACL), above some RMS threshold. This correlation decreases as the material becomes less brittle (i.e., decrease of Young’s modulus or increase of percent of elongation). Simultaneously, a boundary value problem involving traction-free random rough surfaces was solved using a perturbation method, assuming elastic and isotropic conditions. For small RMS/ACL ratio, the solution for the RMS stress concentration factor, kt was found to be: kt = 1 + 2*SQRT(2)*(RMS/ACL), which agrees very well with the experimental work. Finally, a generalization of stress concentration factor formulas for several geometrical configurations and loading conditions into the Modified Inglis Formula was proposed. Finite element analysis was carried out and comparison was made with both experimental and analytical results. Applications of these results are broad. In surface engineering, for example, our analytical solution can be coupled with Fick’s Law to find critical conditions under which a film could become unstable to random roughness. Additionally, in design and maintenance of surfaces in service, it can be used to preliminarily assess how stress concentrates in surfaces where well defined notches cannot be used as an approximation.

Thrombosis is one of the leading causes of mortality and morbidity that is associated with myocardial infarction, stroke and pulmonary embolism. Anti-thrombotic agents which intend to reduce the occurrence and severity of thrombosis usually target the enzymes of the coagulation cascade. FXIa, a 160 kDa homodimer is gaining popularity of late as a potential target for anti-thrombotic agents due to its relative safety. A number of inhibitors which target the active site of FXIa have been reported but to our knowledge there have been no inhibitors which act via an allosteric mechanism. The aim of this project was to screen for allosteric inhibitors of FXIa from of pool of sulfated small-molecules.These molecules were primarily designed to act as heparin mimetics; heparin being a natural anti-coagulant. These compounds were then analyzed to determine whether inhibition was via an allosteric mechanism.

Epithelial to mesenchymal transition occurs in the later stages of epithelial tumor progression, with cells expressing mesenchymal markers. Of these, the intermediate filament protein vimentin is frequently upregulated in metastatic carcinomas. Previously, microarray studies showed that the gene encoding vimentin is highly upregulated in metastatic HN12 cells compared to a related primary tumor cell line. In this study, we confirmed this difference using real-time quantitative PCR, western blot analysis, and immunostaining. Furthermore, EGF and TGF-β, growth factors that induce migration and invasion of HN12 cells, produced synergistic increases in vimentin expression. To assess the contribution of vimentin to the biological properties, HN12 cells were stably transfected with a plasmid that directs synthesis of vimentin shRNA. Clones expressing decreased amounts of vimentin were isolated and characterized. These cells showed significantly reduced proliferation compared to non-targeting controls. Moreover, downregulation of vimentin led to a decrease in cell motility, as well as reducing their ability to invade through a basement membrane substitute. Using transient transfection assays, vimentin promoter activity was determined in HN12 cells to define regulatory elements important for controlling vimentin upregulation in the absence or presence of EGF and TGF-β. Taken together, the data indicate that overexpression of vimentin is important for proliferation and invasion of metastatic HN12 cells, and suggest that EGF- dependent pathways target binding elements in the proximal vimentin promoter, while TGF-β is likely to act in an AP1-dependent manner. Furthermore, both growth factors appear to synergize by stimulating promoter activation through the ASE site, suggesting involvement of Stat-dependent pathways in regulation of vimentin expression in HN12 cells.

Porphyromonas gingivalis, a gram-negative anaerobic, pathogenic bacterium is a major etiological agent in the initiation and progression of periodontal disease. Due to the ever-changing environment of the oral cavity, inhabitants like Porphyromonas gingivalis must possess the ability to adapt to changes in environmental conditions like pH, temperature, oxygen tension, and metal concentration. P. gingivalis should therefore have an efficient regulatory system in order to adapt and survive in the oral cavity. This response adaptation occurs at the transcriptional level, which involves alternative sigma factors. Extracytoplasmic function sigma (ECF-s) factors are the largest group of alternative sigma factors that play a role in the bacterial response to environmental stress conditions. Here we analyze the s-70 factor gene, PG0214, an extracytoplasmic function sigma factor encoded in the P. gingivalis genome, and examine its role in the bacterial response to environmental stress and virulence. Our findings indicate that the PG0214 gene is important in regulating major functional gene groups and pathways in the P. gingivalis genome. Strains deficient in the PG0214 gene were analyzed and shown to have decreased protease activity, as well as reduced survivability and invasion rates in eukaryotic host cells when compared against wild-type W83 and ATCC 33277 strains. Collectively our studies demonstrate that the PG0214 gene is a positive regulator of gene expression for the survival and virulence of P. gingivalis in the presence of oxidative- and iron-stress, although further study is needed to fully characterize the gene and determine its specific function.

Articular cartilage is an extraordinary tissue, allowing frictionless movements of articulated joints, and acting as a load-bearing cushion to protect joints from damage. Breakdown of articular cartilage may result in crippling diseases such as osteoarthritis (OA) and, since articular cartilage has a limited repair capacity, a greater understanding of the mechanisms of joint homeostasis and its response to injury are of great clinical need. In this project the hypothesis that endogenous mesenchymal stem cells (MSCs) may contribute to the healing process of a full-thickness articular cartilage defect was investigated by combining a mouse model of joint surface injury and repair with a nucleoside analogue labelling scheme in DBA/1 mice. Following injury, proliferative responses of nucleoside analogue-retaining cells were detected between 4 and 12 days post injury (dpi) in both the bone marrow and the synovial membrane of the knee joint. Phenotypic analysis of these label-retaining cells using immunofluorescence staining revealed an MSC-compatible phenotype (CD44+, CD105+, CD146+, PDGFRα+ and p75NGFR+), with differences observed between the two tissues in expression of CD105 and CD146. The response of the label-retaining cells to the injury was associated with early activation of Notch signalling (4 dpi), followed by BMP signalling at 8 dpi and TGF-β at 12 dpi. Conversely, canonical Wnt signalling, which was active in uninjured knee joints and in injured knee joints up to 8 dpi, was attenuated at 12 dpi. The contribution of nerve growth factor (NGF), known as a pain mediator in OA, to the repair process was then investigated in vitro. NGF was released by both cartilage explants and femoral head cultures following injury. Using a Transwell-based cell migration assay, NGF was revealed to have a chemotactic effect on human bone marrow derived MSCs, but not synovial membrane derived MSCs. High-density micromass cultures also revealed NGF had a potent stimulatory effect on the chondrogenic differentiation of mesenchymal cells. The data presented here demonstrate a contribution of endogenous MSCs to the repair of articular cartilage in vivo and suggest a possible new therapeutic strategy: stimulation of in vivo recruitment of MSCs by modulating signalling pathways activated during the healing process. Furthermore, a novel role for NGF as a factor involved in migration and the chondrogenic differentiation of MSCs is suggested.

3 ABSTRACT Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of biophysics and physical chemistry Author: Magdaléna Pleváková Supervisor: Ing. Vladimír Kubíček, CSc. Thesis title: Determination of Lipophilicity of Drugs by HPLC In this thesis a RP-HPLC method for fast and reliable determination of lipophilicity was proposed and tested. Stationary phase was selected by using hydrophobic substraction model. Capacity factors of the chosen substances were initially measured on Zorbax ECLIPSE XDB C18 250x4,6 mm, 5µm column, which exhibits almost identical retention characteristics as the column used for this purpose until now. Then the capacity factors of the same substances were determined by using Zorbax ECLIPSE XDB-C18 50x4,6 mm, 1,8µm column that was selected to reduce retention times significantly. A group of newly synthesised drugs based on structure of pyrazine served as samples for the measurement. The reproducibility of the capacity factor values determined using both columns was compared and the independence of the capacity factor on the mobile phase flow was confirmed. The capacity factors of two homologous series and a group of benzimidazols were consequently determined on Zorbax ECLIPSE XDB-C18 50x4,6 mm, 1,8µm column using various compositions of mobile phases. Several...